myCAF-derived exosomal PWAR6 accelerates CRC liver metastasis via altering glutamine availability and NK cell function in the tumor microenvironment

Abstract

Background: Liver metastasis from colorectal cancer (CRC) is a major clinical challenge that severely affects patient survival. myofibroblastic cancer-associated fibroblasts (myCAFs) are a major component of the CRC tumor microenvironment, where they contribute to tumor progression and metastasis through exosomes.

Methods: Single-cell analysis highlighted a notable increase in myCAFs in colorectal cancer liver metastases (CRLM). Exosomal sequencing identified PWAR6 as the most significantly elevated lncRNA in these metastatic tissues. In vivo and in vitro assays confirmed PWAR6's roles in CRC cell stemness, migration, and glutamine uptake. RNA pulldown, RIP, and Co-IP assays investigated the molecular mechanisms of the PWAR6/NRF2/SLC38A2 signaling axis in CRC progression, flow cytometry was used to assess NK cell activity and cytotoxicity.

Results: Clinically, higher PWAR6 expression levels are strongly associated with increased ⁶⁸Ga FAPI-PET/CT SUVmax values, particularly in CRLM patients, where expression significantly exceeds that of non-LM cases and normal colon tissues. Regression analysis and survival data further support PWAR6 as a negative prognostic marker, with elevated levels correlating with worse patient outcomes. Mechanistically, PWAR6 promotes immune evasion by inhibiting NRF2 degradation through competitive binding with Keap1, thereby upregulating SLC38A2 expression, which enhances glutamine uptake in CRC cells and depletes glutamine availability for NK cells.

Conclusion: myCAFs derived exosomes PWAR6 represents a pivotal marker for CRC liver metastasis, and its targeted inhibition with ASO-PWAR6, in combination with FAPI treatment, effectively curtails metastasis in preclinical models, offering promising therapeutic potential for clinical management.

Fig. 1

myCAFs enhanced CRC cell stemness and promote migration. A Uniform manifold approximation and projection (UMAP)plot showing the eight major cell types. Dots represent individual cells and colors represent different cell populations. (n = 18 patients). B Single cell data clustering analysis, data from GSE16655 and GSE178318. (n = 18 patients). C Proportion of patients with different Stages in high myCAF and low myCAF score groups, data obtained from the metaGEO database. (n = 913 patients). D Kaplan‒Meier survival curve of patients with high myCAF score and low myCAF score, data obtained from the metaGEO database. (n = 913 patients). E Workflows of the procedure for extracting myCAF and PDOs from CRC Patients and assessing invasion capability after co-culture with CRC Cells. F Western blot analysis of α-SMA, FAP and Collagen I protein expressions in NFs and myCAFs isolated from CRC patients. (myCAF and NF were isolated from four patients). G Immunofluorescence analysis of α-SMA, FAP and Collagen I protein expressions in NFs and myCAFs isolated from CRC patients. Scale bars = 100μm (left), Scale bars = 20μm (right). (n = 3, one of three biological replicates). H Representative α-SMA staining in CRC tissues from FUSCC cohort 2. Scale bars = 50μm. (n = 3, one of three biological replicates). I, J Transwell assay to evaluate the invasion after co-cultured DLD1 with myCAFs. Scale bars = 100μm. (Representative images are shown, data are from five biologically replicates). K, L Sphere forming assay to evaluate the stemness after co-cultured DLD1 with myCAFs. Scale bars = 100μm. (Representative images are shown, data are from five biologically replicates). M, N PDOs 3D invasion assay to evaluate the invasion of PDOs after co-cultured PDOs with myCAFs. Scale bars = 50μm. (Representative images are shown, data are from three replicates). O, P Representative images of subcutaneous tumors treated with FAPI gavage. (n = 5 mice per group). Q, R Liver metastasis assays were performed in vivo by splenic injection to evaluate the effect of FAPI on tumor metastasis. (n = 5 mice per group). Data are shown as mean ± s.e.m. For B-C, data were analyzed by chi-square test. For D, data were analyzed by log-rank test. For I-R, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

myCAF-derived exosome PWAR6 enhanced glutamine uptake and promote CRC cell migration. A–C Evaluation of invasion and stemness of DLD1 Cells after GW4869 treatment using Transwell and spheroid formation assays. Scale bars = 100μm(upper), Scale bars = 50μm(lower). (Representative images are shown, data are from five biologically replicates). D PDO 3D invasion assay to evaluated the invasion of PDOs after GW4869 treatment. Scale bars = 50μm. (Representative images are shown, data are from three biologically replicates). E Workflows of the extraction of fibroblasts from LM tissues (myCAFs) and adjacent normal tissues (NFs) in CRLM patients, followed by the extraction of exosomes for lncRNA sequencing and metabolomics analysis. F Expression of the exosome markers TSG101, HSP70 and Alix confirmed by western blot. (n = 3, one of three biological replicates). G Transmission electron microscopy (TEM) images of exosomes secreted from myCAFs and NFs. Scale bars = 100nm. (n = 3, one of three biological replicates). H Top 100 upregulated and downregulated lncRNAs in exosomes secreted by myCAFs from CRLM tissues and paired NFs. I The expression levels of PWAR6 among normal colonic epithelial cell line (NCM460), CRC cell lines, CRC tissues (T), normal intestinal epithelial tissues (N), NFs, NF-derived exosomes, myCAFs, and myCAF-derived exosomes. (n = 3 technical replicates, one of three biological replicates). J ISH experiments in CRC cells and PDO indicate that PWAR6 is localized in both the nucleus and the cytoplasm. Scale bars = 100μm. (n = 3, one of three biological replicates). K Knockdown of PWAR6 in myCAFs. (n = 5, one of three biological replicates). L Knockdown of PWAR6 in myCAFs reduces its level in exosomes secreted by myCAFs. (n = 5, one of three biological replicates). M Incubation with exosomes from PWAR6-knockdown myCAFs significantly reduces PWAR6 levels in DLD1 cells. (n = 5, one of three biological replicates). N, O PDO 3D invasion assay to evaluated the invasion of PDOs after knocking down PWAR6. Scale bars = 50μm. (Representative images are shown, data are from three biologically replicates). P, Q PWAR6 knockdown significantly reduces the size of subcutaneous tumors. (n = 5 mice per group). R, S PWAR6 knockdown significantly reduces the size of liver metastases. (n = 3 mice per group). Data are shown as mean ± s.e.m. For A-D, I and K–O, data were analyzed by one-way ANOVA. For P-S, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Expression of exo-PWAR6 and its clinical significance in patients with CRC. A, B Representative image and PWAR6 expression of patients with varied ⁶⁸Ga-FAPI-PET SUV max values (n = 15 patients). C Representative image of PDOs and PWAR6 ISH using tissues from patients with varied ⁶⁸Ga-FAPI-PET SUV max values. Scale bars = 50μm. (n = 15 patients). D, E Representative ISH image and PWAR6 expression in healthy donors (enteritis) and CRC patients with and without LM. Scale bars = 50μm (n = 25 patients). F Representative CT or MRI Images of CRC Patients with and without LM (n = 25 patients). G Heatmap of Chi-Square test based on the association between PWAR6 and different clinicopathological factors. (n = 150 patients). H Univariate and multivariate Cox regression analyses of OS in the FUSCC cohort 3. (n = 150 patients). I PWAR6 expression level of patients with high myCAF score and low myCAF score, data obtained from the GSE39582. (n = 133 patients). K Kaplan‒Meier survival curve of patients with PWAR6-high and PWAR6-low, data obtained from the FUSCC cohort3. (n = 150 patients). Data are shown as mean ± s.e.m. For A-D and I, data were analyzed by one-way ANOVA. For G, data were analyzed by Chi-square test. For H, data were analyzed by Cox regression analysis. For J, data were analyzed by log-rank test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

myCAF-derived exo-PWAR6 enhances the glutamine uptake of CRC cells via upregulation of amino acid transporters SLC38A2. A, B Metabolomics Sequencing of exosomes secreted by myCAFs from CRLM tissues and paired NFs (n = 5 patients). C, D Evaluation of glutamine uptake, glutamate and α-Ketoglutarate in CRC cells co-cultured with myCAFs after knocking down PWAR6. (n = 5, one of three biological replicates). E qRT-PCR to analyze the transcriptional profiling of known glutamine transporters treated with shPWAR6/LvPWAR6 exosomes derived from myCAFs. (n = 3, one of three biological replicates). F, G Western blot to evaluate the protein level of SLC38A2 in DLD1/HCT116 cells treated with myCAF exosomes. (n = 3, one of three biological replicates). H SLC38A2 expression is elevated in metastatic compared to primary tumors, data from GSE41568 (n = 133 patients). I Immunofluorescence of NRF2 and SLC38A2. (n = 3, one of three biological replicates). J, L Evaluation of invasion and stemness of DLD1 Cells after shSLC38A2 treatment using Transwell and spheroid formation assays. Scale bars = 100μm(upper), Scale bars = 50μm(lower). (Representative images are shown, data are from three biologically replicates). M, N Expression of SLC38A2 in FUSCC Cohort 2 and representative IHC staining images. Scale bars = 50μm. (Representative images are shown, n = 25 patients). O, P Expression of SLC38A2 in FUSCC Cohort 3 and representative IHC staining images. Scale bars = 50μm. (Representative images are shown, n = 150 patients). Q IHC Staining of SLC38A2 in PDOs from patients with varied ⁶⁸Ga-FAPI-PET SUVmax. Scale bars = 50μm. (Representative images are shown, n = 15 patients). R, S SLC38A2 knockdown significantly reduces the size of subcutaneous tumors. (n = 5 mice per group). T, U SLC38A2 knockdown significantly reduces the size of liver metastases. (n = 3 mice per group). Data are shown as mean ± s.e.m. For C-E, J-L, data were analyzed by one-way ANOVA. For H, M-U, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

PWAR6 blocks NRF2 ubiquitination and degradation. A Co-culture of CRC cells and myCAFs before pull-down assay. B, C Silver stained SDS-PAGE gel of proteins immunoprecipitated by the sense and antisense of PWAR6. The differentially exhibited lanes were used for the mass spectrum. D Nucleus-cytoplasm separation experiment showed that NRF2 is mainly located in the nucleus. (n = 3, one of three biological replicates). E Representative images of immunofluorescent staining of NRF2 and PWAR6 in DLD1/RKO cells. Scale bars = 50μm. (n = 3, one of three biological replicates). F Western blotting assays (upper part) and RIP assay (lower part) of the specific interaction of PWAR6 with NRF2. (n = 3, one of three biological replicates). G The secondary structure of PWAR6 is shown as predicated by the centroid method (http://rna.tbi.univie.ac.at/). H Truncated PWAR6 fragments interacting with NRF2 in DLD1 cell lysates. (n = 3, one of three biological replicates). I Western blot to evaluate the protein level of NRF2 in DLD1/HCT116 cells treated with myCAF exosomes. (n = 3, one of three biological replicates). J–M Protein stability assay by using cycloheximide (CHX, 50 μg/mL) to treat cells at the different time was performed to evaluate the effect of PWAR6 overexpression (J, K) or knockdown (L, M). (n = 3, one of three biological replicates). N Western blot analysis of NRF2 in DLD1/HCT116 cells treated with myCAF overexpression or knockdown exosomes with the proteasome inhibitor MG132 (10 μM). (n = 3, one of three biological replicates). O, P Co-IP was used to assess NRF2 ubiquitination with and without PWAR6 overexpression (O) or knockdown (P), following MG132 and HA-Ub treatment. (n = 3, one of three biological replicates). Q–T Transwell assay to evaluate the DLD cell invasion (upper part and R), Scale bars = 100μm, spheroid formation assays to evaluate the DLD cell stemness (middle part and S), PDOs 3D invasion assay to evaluate the invasion of PDOs after knockdown of NRF2 (lower part and T). Scale bars = 50μm. (Representative images are shown, data are from biologically replicates). Data are shown as mean ± s.e.m. For F, K, M and Q-T, data were analyzed by one-way ANOVA. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

PWAR6 accelerates migration, stemness and glutamine uptake by elevating the expression of NRF2. A The average intensity curves for NRF2 signals at TSSs in a region comprising ± 2 Kb in DLD1 and HCT116 cell lines. B The IGV shows the CUT&Tag signals of NRF2 at the SLC38A2 promoter. C Heatmap of CUT&Tag-seq peaks associated with the NRF2 in DLD1 and HCT116 cell lines, signals are displayed from −2.0 kb to + 2.0 kb surrounding the TSS. Histone H3 as positive control and IgG as negative control. D The binding sequence of NRF2 within the promoter region of the SLC38A2 gene. E Luciferase assays confirmed that NRF2 regulates SLC38A2 transcription, and PWAR6 overexpression significantly boosts NRF2 activity. (n = 3, one of three biological replicates). F Luciferase reporter assays of the transduced DLD1 cells transfected with reporter plasmids containing the SLC38A2 promoter, respectively. Wild type: −2000–0 construct; mutant: −2000–0 constructed with a point mutation at the NRF2 binding site. (n = 3, one of three biological replicates). G Westen blot of SLC38A2 after NRF2 knockdown. (n = 3, one of three biological replicates). H, I Expression of NRF2 in FUSCC Cohort 2 and representative IHC staining images. Scale bars = 50μm. (Representative images are shown, n = 25 patients). J, K Expression of NRF2 in FUSCC Cohort 3 and representative IHC staining images. Scale bars = 50μm. (Representative images are shown, n = 150 patients). L IHC Staining of NRF2 in PDOs from patients with varied 68Ga-FAPI-PET SUVmax. Scale bars = 50μm. (Representative images are shown, n = 15 patients). M, N NRF2 knockdown significantly reduces the size of subcutaneous tumors. (n = 5 mice per group). O, P NRF2 knockdown significantly reduces the size of liver metastases. (n = 3 mice per group). Data are shown as mean ± s.e.m. For E, data were analyzed by one-way ANOVA. For F, H-P, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

SLC38A2 mediated glutamine deficiency in NK cell promotes tumor migration. A, B Single cell data clustering analysis, data from GSE16655 and GSE178318. (n = 18 patients). C, D The percentage of immune cells in liver tumors from LvPWAR6 mice. (n = 3 technical replicates, one of three biological replicates). E The representative image of NK1.1 staining in subcutaneous tumors. Scale bars = 20μm. (n = 3, one of three biological replicates). F The representative image of NK1.1 staining in liver tumors. Scale bars = 20μm. (n = 3, one of three biological replicates). G Co-culture of NK91MI cells and myCAFs. (NK92MI in the upper chamber, myCAFs in the lower chamber). H, I Representative flow cytometry image and statistical graphs of NK92MI apoptosis in the NK92MI/myCAFs co-culture system. (Representative images are shown, data are from three biologically replicates). J Representative flow cytometry image of NK92MI proliferation in the NK92MI/myCAFs co-culture system. (n = 3, one of three biological replicates). K Co-culture of NK91MI cells, CRC cells and myCAFs. (NK92MI in the upper chamber, CRC cells and myCAFs in the lower chamber). L, M Representative flow cytometry image and statistical graphs of NK92MI apoptosis in the NK92MI/CRC cell/myCAFs co-culture system. (Representative images are shown, data are from three biologically replicates). N Representative flow cytometry image of NK proliferation in the NK92MI/CRC cells/myCAFs co-culture system. (n = 3, one of three biological replicates). O, P Representative images and statistical graphs for CD107a, Granzyme B, and Perforin detection in NK92MI cells. (Representative images are shown, data are from three biologically replicates). Data are shown as mean ± s.e.m. For C-D, H-J, L-P, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

PWAR6 is a potential therapeutic target for CRC. A Graphical diagram of vein tail injection of in vivo optimized PWAR6 inhibitor and FAPI gavage in C57BL/c mice. B–E Representative images of xenograft tumors (B, C) and liver metastasis (D, E) from each mouse group are shown. Mice with xenograft tumors were treated with either an antisense oligonucleotide control (ASO-Ctrl), an in vivo optimized PWAR6 inhibitor (ASO-PWAR6), or a combination of ASO-PWAR6 and FAPI administered via gavage. (n = 5 mice per group). F, G Representative images of NK1.1 and α-SMA IHC staining of xenograft tumors and liver metastasis in each group. Scale bars = 100μm. (n = 3, one of three biological replicates). H, I Representative images of pan-CK, α-SMA, CD3 and CD56 staining in tumor tissues from CRC cancer in FUSCC Cohort2. Scale bars = 50μm. (n = 3, one of three biological replicates). Data are shown as mean ± s.e.m. For B-E, data were analyzed by one-way ANOVA. P values. *P < 0.05, **P < 0.01, ***P < 0.001