Oral Cancer Stem Cell-Derived Small Extracellular Vesicles Promote M2 Macrophage Polarization and Suppress CD4+ T-Cell Activity by Transferring UCA1 and Targeting LAMC2

Cancer-derived small extracellular vesicles (sEVs) are emerging as crucial mediators of intercellular communication between cancer cells and M2-tumor-associated macrophages (M2-TAMs) via transferring lncRNAs. We previously reported that miR-134 blocks the expression of its targeting protein LAMC2 via the PI3K/AKT pathway and inhibits cancer stem cell (CSC) migration and invasion in oral squamous cell carcinoma (OSCC). This study hypothesize that OSCC-CSC-derived small extracellular vesicles (OSCC-CSC-sEVs) transfer a ceRNA of miR-134 and consequently promote M2 macrophage polarization by targeting LAMC2 via the PI3K/AKT pathway through in vitro and in vivo experiment methods. The results showed that sEVs derived from CD133+CD44+ OSCC cells promoted M2 polarization of macrophages by detecting several M2 macrophage markers (CD163, IL-10, Arg-1, and CD206+CD11b+). Mechanistically, we revealed that the lncRNA UCA1, by binding to miR-134, modulated the PI3K/AKT pathway in macrophages via targeting LAMC2. Importantly, OSCC-CSC-sEV transfer of UCA1, by targeting LAMC2, promoted M2 macrophage polarization and inhibited CD4+ T-cell proliferation and IFN-γ production in vitro and in vivo. Functionally, we demonstrated that M2-TAMs, by transferring exosomal UCA and consequently targeting LAMC2, enhanced cell migration and invasion of OSCC in vitro and the tumorigenicity of OSCC xenograft in nude mice. In conclusion, our results indicated that OSCC-CSC-sEV transfer of UCA1 promotes M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis, thus facilitating tumor progression and immunosuppression. Our findings provide a new understanding of OSCC-CSC molecular mechanisms and suggest a potential therapeutic strategy for OSCC through targeting CSC-sEVs and M2-TAMs.


Introduction
The tumor microenvironment provides survival conditions that may enable tumor growth and progression [1]. It is composed of tumor cells and a variety of stromal cells, including matrix immune cells, among which mononuclear cells or macrophages are an abundant and important component [2,3]. Under different regulatory mechanisms, mac-rophages can differentiate into two main subtypes: the classically activated M1 subtype exhibiting antitumor immunity and inflammatory responses and the alternatively activated M2 subtype exhibiting a protumor effect that contributes to tumor development and progression [4,5]. Tumor-associated macrophages (TAMs) are a macrophage population recruited and educated by cancer cells [2,3]. TAMs are typically maintained in an M2-polarized condition with a protumor phenotype involving remodeling of the extracellular matrix, immunosuppression, and tumor progression in the tumor microenvironment [5].
Small extracellular vesicles (sEVs) including exosomes act as nanoscale messengers. sEVs have emerged as crucial mediators of intercellular communication between cancer cells and stromal cells in the tumor microenvironment and have been found to function by transferring cargos, including proteins and RNAs [6,7]. Recently, studies have shown that cancer-derived sEVs promote M2 macrophage polarization through immune signaling pathways by transferring noncoding RNAs in various cancers. For instance, oral cancer-derived exosomes promote M2 macrophage polarization, mediated by exosome-enclosed miR-29a [8]. Cancer stem cells (CSCs), a small subpopulation of cancer cells, can be identified and isolated according to their expression of distinctive markers. CSC-derived-sEVs are reported to be responsible for disease progression of various cancers [9][10][11]. Interestingly, CSCs secrete sEVs associated with an immunosuppressive microenvironment and further promote M2 macrophage polarization in glioblastoma [12] and colon cancer [13]. However, whether CSC-derived sEVs transfer lncRNAs that promote M2 macrophage polarization has rarely been reported.
Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers and remains a major cause of cancer morbidity and mortality worldwide [14]. We previously reported that miR-134 blocks the expression of its targeting protein LAMC2 via the PI3K/AKT signaling pathway and inhibits CSC migration and invasion in OSCC [15]. We have observed that both sEVs and M2 macrophages exert their biological functions via the PI3K/AKT signaling pathway in various cancers [16][17][18][19][20]. Furthermore, recent reports have shown that LAMC2 induces the infiltration of macrophages in lung cancer [21] and that sEVs transfer a miRNA in ovarian cancer via a LAMC2-mediated PI3K/ AKT axis [22]. On the basis of previous studies, we hypothesized that OSCC-CSC-derived sEVs (OSCC-CSC-sEVs) might transfer a ceRNA of miR-134, thereby promoting M2 macrophage polarization by targeting LAMC2 via the PI3K/AKT signaling pathway. As expected, our results revealed that OSCC-CSC-sEVs transferring the lncRNA UCA1 promoted M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis and further modulated immunosuppression, partly by inhibiting CD4 + T-cell proliferation and IFN-γ production. CD133 + CD44 + cells from Cal27 cells were selected as OSCC-CSCs with a magnetic-activated cell sorting system (130-092-545, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). OSCC-CSCs were identified on the basis of immunofluorescence and sphere formation. Cell sorting and immunofluorescence were performed in accordance with the manufacturer's instructions, as described in our previous study [15].
Cells were transduced according to the instructions for lentiviral infection (Supplementary Table S1) in the following groups: overexpression-(oe-) negative control (NC) lentivirus, oe-UCA1 lentivirus, oe-NC lentivirus+sh-NC lentivirus, oe-UCA1 lentivirus+sh-NC lentivirus, and oe-UCA1 lentivirus+sh-LAMC2 lentivirus. Silencing lentivirus particles was packaged by insertion of the core plasmid (PLKO.1) and auxiliary plasmid (RRE, REV, and Vsvg) of the target gene silencing sequence. The overexpression lentivirus was packaged by insertion of the core plasmid (Fugw-GFP and Plx304) and auxiliary plasmid (RRE, REV, and Vsvg) of the cDNA sequence of the target gene. Lentiviruses were purchased from Genomeditech (Shanghai, China). Primer sequences and plasmid construction were performed by Genomeditech. All experimental steps were implemented according to the manufacturer's instructions.

Isolation and
Identification of CSC-Derived sEVs. sEVs were isolated from the supernatants of Cal27-CSCs and Cal27 cells by differential centrifugation. The collected culture supernatant was centrifuged at 300 × g for 10 minutes, at 2000 × g for 10 minutes, and at 10000 × g for 30 minutes. The supernatant was then ultracentrifuged at 110000 × g for 2 hours to obtain the precipitate containing sEVs. The collected sEVs were resuspended in PBS. The precipitate was filtered with a 0.22 filter to remove small cell debris, resuspended in PBS, and then ultracentrifuged again for 2 hours at 110000 × g to remove the PBS. The sEVs were stored at −80°C before use. The above centrifugation was performed at 4°C. The cells used for sEVs isolation were cultured in medium without sEVs and serum (C38010050, VivaCell, Shanghai, China). The morphology of CSC-derived sEVs was verified with a transmission electron microscope (TEM, H-7650, Hitachi Co., Ltd., Tokyo, Japan) and nanoparticle tracking analysis (NTA, NanoSight LM10 Macrophages with different treatments were cocultured with CD4 + T cells (2 × 10 4 cells/well) in six-well plates (3412, Corning Inc., Corning, N. Y., USA). At 24 hours after the macrophages had been seeded in six-well plates, CD4 + T cells were seeded in the transwell chamber above the macrophages. After 48 hours, CD4 + T cells were collected. The CD4 + T cells were fixed and permeabilized with Cytofix/ Cytoperm (88-8823-88, Thermo Fisher Scientific), and the cells were stained with fluorescein-conjugated antibodies to cytokines (PE-interferon-gamma [IFN-γ], 554552, BD Bioscience). The proliferation of CD4 + T cells was detected by the carboxyfluorescein diacetate succinimidyl ester (CFSE, Beyotime) dilution method. CD4 + T cells were then stained with 1 μmol/L CFSE dye and incubated at 37°C for 10 minutes. Dead cells were excluded with a Live/Dead cell staining kit. A flow cytometer (FACSCalibur, BD Bioscience) was used for analysis.

Reverse Transcription-Quantitative Polymerase Chain
Reaction (RT-qPCR). After total RNA was extracted with TRIzol (16096020, Thermo Fisher Scientific), the purity and concentration of the RNA were evaluated according to the absorbance at 260 and 280 nm, measured by spectrophotometry. The A260/A280 ratio of the sample was ≥ 1.8. For mRNA detection, a reverse transcription kit (RR047A, Takara, Kyoto, Japan) was used to reverse transcribe mRNA to obtain cDNA. For miRNA, a Poly(A) Tailing detection kit (B532451, Sangon Biotech, Shanghai, China) was used (including Universal PCR primer R and U6 Universal PCR primer R) and the cDNA of miRNAs containing poly(A) tails was obtained. PCR was performed with a LightCycler 480 instrument and SYBR Green I Master Mix, and the results were normalized to U6 and GAPDH. The 2 −ΔΔCT method was used to determine the ratios of target gene expression between the experimental group and the control group. The primer sequences are shown in Supplementary  Table S2. 2.10. Transwell Assays. A transwell chamber (8 mm aperture; 3422, Corning) was used to detect cell migration in vitro in 24-well plates. First, 600 mL DMEM with 20% FBS was added in the lower chamber and equilibrated at 37°C for 1 hour. Cal27 cells with different treatments were resuspended in DMEM without FBS. Then, 3 × 10 5 cells/mL cells were seeded into the chamber and cultured at 37°C and 5% CO2 for 24 hours. After the transwell insert was removed, the cells in the chamber were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet for 10 minutes. The surface cells were removed with a cotton ball and observed under an inverted fluorescence microscope (TE2000, Nikon, Tokyo, Japan). Five fields of vision were read randomly and photographed. The number of cells that passed through the chamber was counted. The average value was the number of cells passing through the chamber in each group. For cell invasion tests, the transwell chamber was precoated with Matrigel (356234, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) to simulate the cell matrix. Three duplicate wells were prepared for each group.
2.11. Tumor Xenograft Model. All experimental procedures were approved by the Animal Ethics Committee of Shanghai Ninth People's Hospital, in compliance with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). BALB/C adult male nude mice 6 weeks of age were reared under specific-pathogen-free conditions and given free access to drinking and food. The mice were randomly divided into three groups with six mice each: the oe-NC+sh-NC group, the oe-UCA1+sh-NC group, and the oe-UCA1+sh-LAMC2 group. Cal27 cells and macrophages treated with different Cal27-CSC-sEVs were suspended in PBS and implanted into the right axillary subcutaneous tissue in nude mice (1 × 10 6 cells per mouse). Cal27-CSC-sEVs (5 μg/mL) were injected into the caudal vein every 3 days after implantation. At 10 days after Cal27 cell inoculation, T cells (1 × 10 6 cells per mouse) were injected into the peritoneal cavity in the mice. After 28 days, the nude mice were euthanized and the tumor weight and volume were measured. The tumor size was measured with Vernier calipers every 2 days. Tumor volume was calculated from three vertical measurements. After the mice were euthanized with pentobarbital sodium at 50 mg/kg (57-33-0, Shanghai Beizhuo Biotechnology Co., Ltd., Shanghai, China), the tumors were removed and photographed. The spleen was isolated into single cells and analyzed by flow cytometry.
2.12. Statistical Analysis. SPSS 21.0 (IBM Corp. Armonk, NY, USA) was used for statistical analysis. The measurement data are summarized as the mean ± standard deviation. Independent sample t-test was used for comparisons between groups. One-way analysis of variance (ANOVA) was applied for comparisons among multiple groups, and repeated measures ANOVA, followed by Tukey's post hoc test, was used to compare data at different time points. p < 0:05 was considered to indicate a statistically significant difference.

OSCC-CSC-sEVs Promote M2 Polarization of
Macrophages. First, OSCC-CSCs were sorted using magnetic bead sorting technology. Fluorescence microscopy (Figure 1(a)) showed that the sorted CD133 + CD44 + cells emitted red fluorescence and green fluorescence, thus indicating that CD133 and CD44 were highly expressed in OSCC cells. However, no red or green fluorescence was observed in CD133 -CD44cells, thus suggesting that CD133 and CD44 were not expressed in those cells. The results of sphere-forming assays in vitro indicated that the ability of isolated OSCC-CSCs to form spheres was significantly stronger than that of OSCC cells (Figure 1(b)).
Second, OSCC-CSC-sEVs were successfully extracted. OSCC-CSCs were cultured in vitro, and sEVs were isolated from the supernatant. Transmission electron microscopy indicated that the isolated sEVs had typical sEV morphology with a generally consistent round-or oval-shaped membranous vesicle pattern (Figure 1(c)). NTA indicated that the diameters of sEVs ranged from 85 nm to 130 nm (Figure 1(d)). Western blot analysis indicated that the sEV surface markers CD63 and CD9 were highly expressed, but calnexin was not expressed on the surfaces of sEVs (Figure 1(e)).

UCA1
Modulates the LAMC2/PI3K/AKT Axis in OSCC-CSCs via Binding miR-134. We previously reported that miR-134 influenced the biological behavior of OSCC-CSCs via downregulation of the PI3K/AKT signaling pathway and inhibition of LAMC2 expression [15]. lncRNAs are widely recognized as ceRNAs that competitively bind miR-NAs. To identify the lncRNAs that competitively inhibit miR-134 in OSCC, we predicted the lncRNAs that might bind miR-134 according to the RNAInter database and intersected the results with the upregulated lncRNAs in GSE146483; the results indicated that UCA1 binds miR-134 and is highly expressed in OSCC (Figures 2(a)-2(d)). Dual-luciferase reporter assays revealed that a miR-134 mimic significantly decreased the luciferase activity of UCA1-WT, but did not affect the luciferase activity of UCA1-MUT (Figure 2(e)). Hence, UCA1 binds to miR-134. According to the GEPIA database, UCA1 has significantly higher expression in OSCC than normal tissue (Figure 2(f)). In addition, the results of GO and KEGG analyses showed that miR-134 downregulates LAMC2 in OSCC and subsequently regulates the PI3K/AKT pathway (Supplementary Figure S1). According to starBase database analysis, the positive correlation between UCA1 and LAMC2 expression in OSCC was significant (Figure 2(g)). On the basis of these findings and our previous study [15], we speculated that UCA1 might affect the biological behavior of OSCC-CSCs by regulating the LAMC2/PI3K/AKT axis via binding miR-134.

UCA1 Modulates the PI3K/AKT Pathway in
Macrophages via Targeting LAMC2. First, we performed oe-UCA1 treatment in Mφ through lentiviral transduction. RT-qPCR showed that the expression of UCA1, LAMC2, PI3K, and AKT significantly increased, whereas miR-134 expression decreased in Mφ after treatment (Figure 3(a)). Western blot analysis verified that the expression of LAMC2, PI3K, AKT, and p-AKT significantly increased in Mφ after treatment (Figure 3(b)). Second, we performed oe-UCA1 plus sh-LAMC2 treatment in Mφ. The silencing efficiency of sh-LAMC2 was verified by RT-qPCR (Figure 3(c)) and Western blot (Figure 3(d)) analysis; sh-LAMC2-2 was used in subsequent experiments because it had the better silencing effect. RT-qPCR showed that the expression of LAMC2, PI3K, and AKT significantly decreased, whereas the UCA1 and miR-134 expression did not significantly change in Mφ after oe-UCA1 plus sh-LAMC2 treatment (Figure 3(e)). Western blot analysis verified that the expression of LAMC2, PI3K, AKT, and p-AKT significantly decreased after oe-UCA1 plus sh-LAMC2 treatment   Stem Cells International ( Figure 3(f)). Hence, UCA1 modulates the PI3K/AKT pathway via targeting LAMC2 in macrophages.
To further investigate the effect of UCA1 on M2 polarization by targeting LAMC2, we performed oe-UCA1 plus sh-LAMC2 treatment in Cal27-CSC-sEVs cocultured with Mφ. RT-qPCR showed that the expression of CD163, IL-10, and Arg-1 significantly increased after oe-UCA1 treatment, but these effects were negated by additional sh-LAMC2 treatment (Figure 4(f)). Consistently, flow cytometry assays showed that the proportion of CD206 + CD11b + cells was significantly elevated after oe-UCA1 treatment, but this increase was abrogated by additional sh-LAMC2 treatment (Figure 4(g)).
We further investigated the LAMC2-mediated effects of UCA1, delivered by OSCC-CSC-sEVs, on the biological function of OSCC Cal27 cells and Mφ. Differently treated Mφ was cocultured with Cal27 cells. Transwell assays revealed that the migration and invasion of Cal27 cells were enhanced after oe-UCA1 treatment, but these effects were nullified by additional sh-LAMC2 treatment (Figure 4(i)). Differently treated Mφ was cocultured with CD4 + T cells. Flow cytometry assays revealed that the proliferative number 9 Stem Cells International of CD4 + T cells and the proportion of IFN-γ + CD4 + T cells significantly decreased after oe-UCA1 treatment, and this response was augmented by the addition of sh-LAMC2 treatment (Figure 4(h)).  13 Stem Cells International treatment with oe-UCA1, but this response was counteracted by additional sh-LAMC2 treatment. Therefore, OSCC-CSC-sEVs promote macrophage M2 polarization by transferring UCA1 and targeting LAMC2, thus enhancing the tumorigenicity and immunosuppression of OSCC in nude mice. Together, our results revealed that OSCC-CSC-sEVs transferring the lncRNA UCA1 promote M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis and further modulate immunosuppression, partly by inhibiting CD4+ T-cell proliferation and IFN-γ production ( Figure 6).

Discussion
TAMs and associated sEVs play important roles in mediating intercellular communication, modulating immunosuppression, and facilitating cancer progression in the tumor microenvironment by transferring RNAs [3,6]. Earlier studies have investigated cancer cell-derived sEVs, which promote M2 macrophage polarization in various cancers by transferring noncoding RNAs [6][7][8]. However, whether CSC-sEVs transfer noncoding RNAs, thus inducing M2 macrophage polarization, was largely unknown in most cancers [9]. In this study, we revealed that OSCC-CSC-sEVs transferring the lncRNA UCA1 promote M2 macrophage polarization via a LAMC2-mediated PI3K/AKT axis, thereby facilitating tumor progression and immunosuppression.
The lncRNA UCA1 has been reported to be involved in malignant progression of OSCC by targeting miR-143 [23] and miR-124 [24]. Furthermore, pancreatic cancer-derived exosomal UCA1 has been found to promote tumor angiogenesis by targeting miR-96 [25]; exosomal UCA1 modulates cervical cancer stem cell self-renewal and differentiation by targeting miR-122 [26]. However, the association between UCA1 and TAMs in OSCC was unknown. We found that UCA1, delivered by OSCC-CSC-sEVs, contributed to the migration and invasion of OSCC by binding to miR-134. Moreover, several reports have shown that OSCC-derived exosomes, by transferring proteins and miRNAs, promote M2 macrophage polarization [ [8]. However, the association between UCA1 and macrophages in OSCC was unknown. We demonstrated that UCA1 delivered by OSCC-CSC-sEVs promotes M2 macrophage polarization.
M2-tumor-associated macrophages (M2-TAMs) are among the most abundant immunosuppressive cell types in the tumor microenvironment. M2 macrophage polarization further contributes to immunosuppression via inhibiting T-cell proliferation and the production of relevant cytokines [31][32][33]. In this study, we revealed that OSCC-CSC-derived exosomal UCA1, by targeting LAMC2, promotes M2 macrophage polarization and inhibits CD4 + Tcell proliferation and IFN-γ production in vitro and in vivo. Importantly, we demonstrated that M2-TAMs, by transferring exosomal UCA1 and targeting LAMC2, enhance cell migration and invasion of OSCC in vitro and the tumorigenicity of OSCC in nude mice in vivo. Furthermore, increasing evidence indicates that tumor-derived 14 Stem Cells International exosomes and M2-TAMs play roles in the malignant biological behavior of OSCC [34,35]. However, their immunosuppressive mechanisms and functional roles in OSCC microenvironment remain to be further explored [2].

Conclusions
In summary, our data suggested that the lncRNA UCA1 increased in OSCC-CSC-derived sEVs, and these UCA1-rich CSC-secreted sEVs were transferred to unpolarized macrophages and induce macrophage polarization toward protumor-related M2 macrophages by targeting LAMC2 via the PI3K/AKT pathway. These findings indicated that the OSCC-CSCs use sEV-transferring UCA1 to modulate the immunosuppressive microenvironment, thus enabling cell migration and invasion of OSCC and enhancing tumorigenicity. Our findings provide a new understanding of the molecular mechanism of OSCC-CSC and suggest a potential therapeutic strategy for OSCC by targeting CSC-sEVs and M2-TAMs.

Data Availability
The data used to support the findings of this study are included within the article.