3.1 Identification of SPINT3 as a possible metastatic suppressor by using CRISPR knock out sub-library
TGF-β1 is initially consisted of ∼50-kDa pro-protein containing growth factor, LAP and TGF-β binding protein such as LTBP1(latent transforming growth factor β binding proteins 1), LTBP2 . Once the TGF-β1 binding protein is deficient, secretion of TGF-β1 would be highly increased, which would further induce the tumor growth and metastasis .
In light of the important role of TGF-β binding protein in cancer, we first extracted 22 genes from the transforming growth factor beta binding pathway (GO:0050431), and then synthesized an sgRNA library to target these 22 genes, as shown in Fig. 1A. After 12 weeks, all the SCID mice were dissected, and the presence of the GFP signal was detected. Among the 20 SCID mice implanted with gastric cancer tissue from the sgRNA library group, 12 developed lung metastasis, and the other 8 SCID mice implanted with MKN-45 primary tumor tissue did not have any metastatic tumors, suggesting that some of the driver gene in metastasis was knock out in those 12 mice(Fig. 1B). We harvested the lung tissue from those 12 metastatic library mice, and extract the genome DNA, then amplified by the sgRNAs specific primers as descripted in the methods 2.5 part, we confirmed that SPINT3 sgRNA showed up in every mice belong to the metastatic library mice (Fig. 1C) other than the non-metastatic library mice (Fig. 1G). also for the other 21 genes, VASN sgRNA was present in 6 mice from metastatic library mice, and TWSG1 sgRNAs was present in 5 mice from metastatic library mice (Fig. 1C, D). VASN gene is tumor related gene which already been studied. In lung cancer, VASN is a potential lung cancer–promoting factor, overexpression of VASN could promote the bronchial epithelial cell transformation through autophagy-mediated apoptosis . In glioma cancer, VASN is responsible for the maintenance of glioma stem-like cells through the regulation of NOTCH1 signaling , as to gastric cancer, it’s already been studied in a PhD thesis even it’s not been published (DOI:10.27232/d.cnki.gnchu.2021.000019). And the relation between TWSG1 and gastric cancer already been proved, TWSG1 could bound to BMP4 and inhibited BMP4-binding activity to BMPR-II , it could also been a target of miR-593-5p, and further modulate the proliferation of gastric cancer cell in vivo and in vitro . Thus we preliminary chose SPINT3 for further investigation due to the high frequency of SPINT3 sgRNA in metastatic tissue, also to avoid the conflicts with existing research.
As to the other 8 non-metastatic mice, they were sacrificed at the same time to verify the metastasis. Unlike the clinical situation, which the most common sites of metastasis in gastric cancer patients were liver and peritoneum, the liver of all the metastatic library mice were normal (Fig. 1E), in the meantime, all the sgRNAs target 22 genes such as SPINT3 did not showed up in the liver and peritoneum tissue of metastatic library mice (Fig. 1F), suggesting the mouse model we developed mainly focus on the lung metastasis. Furthermore, by using QPCR, we proved that the expression of SPINT3 in mRNA level is lower in gastric cancer tissue, lung metastatic tissue from metastatic library mice compared with non-metastatic mice, which indicated that the SPINT3 low expression cell had highly metastasis ability (Fig. 1H). Based on these results, we chose SPINT3 as our candidate gene for further analysis.
3.2 Deficiency of SPINT3 increased lung metastasis of gastric cancer cell line
According to the literature, the function of SPINT3 includes serine-type endopeptidase inhibitor activity ; however, the exact role of SPINT3 in gastric cancer remains unknown. Hence, we first downloaded the gastric cancer dataset from the TCGA database, there are 35 healthy donor tissue and 415 gastric cancer tissue included in this dataset, we then extract the RSEM value of SPINT3 and normalized by log2 transformation, and confirmed that expression SPINT3 in these 2 groups were 9.51 ± 3.16 vs 4.38 ± 2.19, using 2-tailed t-test, we confirmed that SPINT3 expression was significantly lower in gastric cancer tissue than in healthy tissue ( P < 0.05) (Fig. 2A). We also detect the expression of SPINT3 in 5 pairs of tumor adjacent tissue and gastric cancer tissue, and confirmed that the expression SPINT3 in mRNA level is 1.09 ± 0.23 vs 0.31 ± 0.16, P value is lower than 0.0001(Fig. 2B). Among these 5 pairs of tissue, we selected 3 pairs as there was not enough tissue in the other 2 pairs. By using western blot, we also proved the consistently low expression of SPINT3 in stomach tumor tissue (Fig. 2C). From a clinical perspective, TCGA-STAD dataset were divided into SPINT3 low and SPINT3 high group based on the median of RSEM value of SPINT3, then using univariate COX to assess the difference of overall survival rate, patients with low SPINT3 expression would have a poorer prognosis, P value lower than 0.05, and HR (95% CI for HR) is 0.82(0.59–0.96), suggesting that SPINT3 is a possible tumor suppressor in gastric cancer (Fig. 2D).
To further validate the role of SPINT3 in gastric cancer, we then chose gastric cancer cell line MKN-45 and MFC. For the MFC cell line, it’s also been called as CVCL_5J48 and derived from 615 mice, similar to the mouse model we developed in Fig. 1A, MFC cells prone to blood-born metastasis to lung in 615 mice, better yet the immune system of 615 mice is normal compared with SCID and nude mice, which perfectly for the subsequently experiment. Given that SPINT3 was consistently expressed at low levels in gastric cancer tissue, we next inhibited SPINT3 by using shRNA and sgRNA approach. Firstly, we set up the MKN-45 CON, MKN-45 SPINT3 sgRNA, MKN-45 SPINT3 shRNA, MFC CON, MFC Spint3 sgRNA, MFC Spint3 shRNA phenotype, and transfected with CON vector (Lenti V2 vector), human SPINT3 sgRNA, human SPITN3 shRNA, mouse Spint3 sgRNA, mouse Spint3 shRNA for 48h, and using western blot to check the expression of SPINT3 in protein level. (Fig. 2E). By using the mouse model developed in Fig. 1A, lung metastasis nodules in SPINT3 sgRNA group was significantly increased compared with the CON group (Fig. 2F) (n = 6 mice/group). We then transformed the fluorescent image to gray scale image, and using Image J software to calculate the change of gray value in CON and SPINT3 KO group, results showed that GFP intensity in SPINT3 sgRNA group was highly increased (Fig. 2G). In addition, lung metastatic tumor tissue from 2 groups were harvested, and using western blot, we detected decreased SPINT3 expression in the lung metastasis nodules (Fig. 2H), proving the link between low expression of SPINT3 and increased lung metastasis.
3.3 Relation between expression of SPINT3 and biological characteristics of gastric cancer cell.
We then designed human and mouse SPINT3 overexpression vector, and transfected it into these 2 gastric cancer lines. Using western blot, we confirmed that the level of SPINT3 expression was elevated in the SPINT3 cDNA-transfected MKN-45 (Fig. 3A upper panel) and MFC (Fig. 3A lower panel). Using Annexin V/PI double staining, we found that apoptosis was induced after transfection of SPINT3 cDNA for 48 h (Fig. 3B), in this process, cleaved caspase 3 was increased, suggesting that the SPINT3 induced apoptosis is caspase dependent (Fig. 3C); moreover, cell proliferation of SPINT3-overexpressing cells in 48 h already been significantly low than CON cells (Fig. 3D), cell migration was also inhibited after transfection of SPINT3 cDNA (Fig. 3E, F). These results indicated that inhibition of SPINT3 was critical for the survival and migration of gastric cancer cell lines.
3.4 Inhibition of TGF-β1/SMAD pathway by the expression SPINT3
Considering the importance of SPINT3 paly in gastric cancer cell proliferation and migration, we then investigate the function of SPINT3. SPINT3 belong to the TGF-β1 binding pathway, even the research about SPINT3 is limited and mainly stayed on the proteasome inhibit, we checked the rest of the genes in TGF-β1 binding pathway, take the LTBP4 as example, it could be secreted by cultured human lung fibroblasts and stored in the ECM(extracellular matrix) either formed the complex with TGF-β1 or as a free form . Deficiency of LTBP4 would release the TGF-β1 and promote tumor metastasis , this inspired us to use in silico analysis to find out the mechanism of SPINT3 induced metastasis. Firstly in the TCGA-STAD dataset, we split gastric cancer patients into ( n = 415 total patients) SPINT3 low expression patients (n = 212 patients) and SPINT3 high expression patients (n = 212 patients), and calculate the up-regulated genes(adjust P value < 0.05, logFC > 1) from SPINT3 low expression group, then using biological process in GO enrichment function, we found that the TGF-β1-related pathway was highly enriched in SPINT3 low expression patients (Fig. 4A). We also roughly check the metastasis-related pathways including PI3K/AKT1, MEK1/GSK3β, β-Catenin and ERK1/2, results showed that most of these pathways were unchanged after SPINT3 is overexpression in MKN-45 and MFC cell lines (Fig. 4B). On the other hand, expression of matured TGF-β1 in MKN-45 was inhibited after the transfection of SPINT3 cDNA, along with the TGF-β1, TGF-β1 downstream component TGF-β1 type1 receptor, TGF-β1 type2 receptor, phosphorylated SMAD3(S423 + S425), total SMAD3 were all suppressed (Fig. 4C), and for the SMAD3, phosphorylated SMAD3 increased more than the total SMAD3 (Fig. 4D). Finally, we applied ELISA to detect the change of total TGF-β1 and active form of TGF-β1, and confirmed that both of them was highly increased in the supernatant of cultured medium, phenotype including MKN-45 CON, MKN-45 SPINT3 sgRNA, MKN-45 SPINT3 shRNA, MFC CON, MFC Spint3 sgRNA and MFC Spint3 shRNA cells, astonishingly that compared with sgRNA group, secretion of total and active TGF-β1 was more in the shRNA group, thus we chose shRNA phenotype for the further experiment (Fig. 4E). Combine these results together, deficiency of SPINT3 might modify the tumor microenvironment via the secretion of TGF-β1.
3.5 Modification of tumor microenvironment by knockout of SPINT3
In the previous section, we stated that knocking out or knocking down SPINT3 could increase the secretion of TGF-β1. TGF-β1 is a well-known factor that regulates cell proliferation , EMT , and cancer metastasis , as well as the tumor microenvironment . In the TGF-β-rich tumor microenvironment, CD8 + T cell proliferation and maturation of T helper cells are inhibited . In our study, to determine the changes in immune cells in the primary tumor, we chose 615 mice owing to their immune system is normal compared with the nude mice and SCID mice we used in the Fig. 1. Firstly, we infected the MFC cell line with the CON or SPINT3 shRNA lentivirus, and injected 5 millions of CON and SPINT3 shRNA MFC cells into 615 mice subcutaneously. Consistent with our in vitro results, knock down of SPINT3 increased tumor cell proliferation in vivo after 2 weeks (Fig. 5A). Then, we sort out the single cell from the primary tumor, and then labeled with different immune cell markers including LY6G, CD8A, CD45, CD11B, CD28, CD3E, LY6C, CD19, CD24, CD14, F4_80, CD326 antibodies, then samples were subjected to CyTOF analysis and clustered, visualized by TSNE, results showed that after SPINT3 was knocked down, CD8 + CD28 + T cells decreased in number, whereas M2 macrophages, PMN-MDSCs (CD11b+Ly6G+Ly6Clow/int), and MO-MDSCs (CD11b+Ly6G−Ly6Chigh) increased in number (Fig. 5B, C). We further validated this change using FACS, consistent with out Cytof results, the number of CD8 + T cells was decreased (Fig. 5D) and the number of PMN-MDSCs (LY6G+) and MO-MDSCs (LY6C+) were increased(Fig. 5E). Moreover, the levels of active markers of MDSCs, including NOS2 and ARG1, significantly increased in SPINT3 knockout tissues (Fig. 5F).
MDSCs are a bone marrow-derived heterogeneous cell population with immunosuppressive activity , and recent studies have shown that the expansion of MDSCs is capable of inhibiting T cell proliferation in vivo and in vitro . Therefore, in our experiment, we sorted MDSCs from the primary tumors in the CON and SPINT3 knock down groups, co-cultured them with T cells from the spleen of 615 mice, and found that the activity of the MDSCs was higher in the SPINT3 shRNA group than in the CON group; the activity was quantified using the percentage of amplified T cells (Fig. 5G). These results suggested that, in SPINT3 knock down primary tumors, the anti-tumor immune response was compromised, reducing cancer cell recognition and clearance, and promoting tumor cell invasion.
3.6 Blockage of SPINT3 related metastasis by reversing the expression of SPINT3 using drug treatment approach.
To block the tumor metastasis bring by SPINT3 inhibition, we firstly trying to reverse the expression of SPINT3 by using drug treatment approach. We check the drug we have in the lab, and found that Apatinib could reverse the expression of SPINT3. Apatinib is a potent inhibitor of VEGF signaling pathway , which could be serve as the downstream pathway of TGF-β1 . In the treatment of gastric cancer, Apatinib is a safe and effective drug for advanced gastric cancer patients . As the function of SPINT3 is tightly linked to TGF-β1, we also selected TGF-β1 inhibitor SB431542 as the candidate, SB431542 is a potent and selective inhibitor of TGF-β1 type 1 receptor , also SB431542 could regulate the secretion of TGF-β1 in gastric , lung cancer cell .
MFC SPINT3 shRNA cells were treated with 25 uM of Apatinib or 1 uM of SB431542 for 72 h, and using western blot, we confirmed that the expression of SPINT3 was elevated by treatment with Apatinib and SB431542(Fig. 6A). Using Annexin FITC/PI double staining and CCK8 experiment, we also concluded that both the Apatinib and SB431542 could induce the apoptosis after 72 h of treatment, also the cell proliferation was inhibition after 48h of treatment (Fig. 6B, C), combined these results together, it’s promising to use Apatinib and SB431542 to stop the SPINT3 related metastasis. So next we injected 5 million of MFC SPINT3 shRNA cells into 615 mice subcutaneously. After 2 weeks of injection, we harvested the primary tumor from mice and digested as single cells, then using the FACS, the decreased percentage of PMN-MDSCs and MO-MDSCs were detected (Fig. 6D). We also test the function of MDSC by using the T cell proliferation assay, results showed that unlike the activated MDSCs sort from primary tumor of SPITN3 shRNA group, activation of the MDSCs from the Apatinib and SB431542 was shut down (Fig. 6E). Using the mouse model in Fig. 1A, we further proved that treatment of Apatinib and SB431542 in vivo could block the metastasis induced by SPITN3 inhibition (Fig. 6F). Combined these results together, we concluded that the gastric cancer metastasis in SPINT3 inhibited tissue could be generally blocked by reversing the expression of SPITN3.
In summary, we discovered the model as shown in Figue 6G, in the SPINT3 deficient tissue, secretion of TGF-β1 as elevated, which activate the function of MDSCs, and inhibit the proliferation of CD8 + T cells, these changes formed the immune suppressed TME, and finally induced tumor metastasis.