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DCLK1 Suppresses Tumor-Specific Cytotoxic T Lymphocyte Function Through Recruitment of MDSCs via the CXCL1-CXCR2 Axis

  • Author Footnotes
    ∗ Authors share co-first authorship.
    Rui Yan
    Footnotes
    ∗ Authors share co-first authorship.
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Author Footnotes
    ∗ Authors share co-first authorship.
    Jianjian Li
    Footnotes
    ∗ Authors share co-first authorship.
    Affiliations
    Institute for Immunology, Department of Basic Medical Sciences, School of Medicine, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China
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  • Zeru Xiao
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Xiaona Fan
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Heshu Liu
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Ying Xu
    Affiliations
    Institute for Immunology, Department of Basic Medical Sciences, School of Medicine, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China
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  • Ruya Sun
    Affiliations
    Institute for Immunology, Department of Basic Medical Sciences, School of Medicine, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China
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  • Jian Liu
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Jiannan Yao
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Guangyu An
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Yan Shi
    Affiliations
    Institute for Immunology, Department of Basic Medical Sciences, School of Medicine, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

    Institute for Immunology, Beijing Key Lab for Immunological Research on Chronic Diseases, School of Medicine, Tsinghua University, Beijing, China
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  • Yang Ge
    Correspondence
    Correspondence Address correspondence to: Yang Ge, MD, Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, 100020 China. fax: (xx) xxx-xxxx.
    Affiliations
    Beijing Chao-Yang Hospital, Department of Oncology, Capital Medical University, Beijing, China
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  • Author Footnotes
    ∗ Authors share co-first authorship.
Open AccessPublished:October 26, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.10.013

      Background & Aims

      Gastrointestinal cancer stem cell marker doublecortin-like kinase (DCLK1) is strongly associated with poor outcomes in colorectal cancer (CRC). Although DCLK1’s regulatory effect on the tumor immune microenvironment has been hypothesized, its mode of action has not been shown previously in vivo, which hampers the potential intervention based on this molecule for clinical practice.

      Methods

      To define the immunomodulatory mechanisms of DCLK1 in vivo, we generated DCLK1-/- tumor cells by CRISPR-Cas9 and developed subcutaneous and intestinal orthotopic transplantation tumor models. Tumor tissues were harvested and subjected to immunofluorescence staining, flow cytometry analysis of tumor-infiltrating immune cell populations, tumor myeloid-derived suppressor cell (MDSC) sorting by isolation kit and then co-culture with spleen T cells, and RNA sequencing for transcriptomic analysis.

      Results

      We found that DCLK1-/- tumor cells lose their tumorigenicity under immune surveillance. Failed tumor establishment of DCLK1-/- was associated with an increase in infiltration of CD8+ T cells and effector CD4+ T cells, and reduced numbers of MDSCs in the tumor tissue. Furthermore, DCLK1 promoted the up-regulation of C-X-C motif ligand 1, which recruits MDSCs in CRC through chemokine C-X-C motif receptor 2. The ability of in vivo tumor growth of DCLK1-/- tumor cells was rescued by C-X-C motif ligand 1 overexpression. Collectively, we validated that DCLK1 promotes tumor growth in CRC through recruitment of T-cell–suppressive MDSCs.

      Conclusions

      DCLK1-mediated immune suppression in tumor models allows escape from the host’s antitumor response. Because DCLK1 is one of the most common markers in gastrointestinal tumors, these results identify a precise therapeutic target for related clinical interventions.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      CFSE (carboxyfluorescein succinimidyl ester), c-Myc (_______), COAD (colon adenocarcinoma), CRC (colorectal cancer), CRISPR/Cas9 (_____________), CSC (cancer stem cell), CXCL (C-X-C motif ligand), CXCR2 (chemokine C-X-C motif receptor 2), DCLK1 (doublecortin-like kinase 1), ELISA (enzyme-linked immunosorbent assay), ERK (extracellular signal-regulated kinase), FBS (fetal bovine serum), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), G-MDSC (granulocytic myeloid-derived suppressor cell), GO (Gene Ontology), IL (interleukin), ICB (immune checkpoint blockade), KEGG (__________), Ki-67 (_____), MAPK (mitogen-activated protein kinase), MDSC (myeloid-derived suppressor cell), MSS (microsatellite stable), NK (natural killer), PBS (phosphate-buffered saline), RNA-seq (RNA sequencing), RT-PCR (reverse-transcription polymerase chain reaction), SC (subcutaneously), TCGA (The Cancer Genome Atlas), TGF-β (tumor growth factor), TME (tumor microenvironment), WT (wild-type)
      High expression of doublecortin-like kinase 1 (DCLK1) in intestinal cancer suggests a poor prognosis. DCLK1 promotes tumor immune evasion through C-X-C motif ligand 1–mediated myeloid-derived suppressor cell recruitment. Abolishing myeloid-derived suppressor cell infiltration by chemokine C-X-C motif receptor 2 blockade eliminates the immune privilege of DCLK1+ tumor and leads to tumor regression.
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      Results

      DCLK1 Knockout Leads to Elimination of Colorectal Cancer Cells in Immune-Competent Hosts

      To explore the role of DCLK1 in tumor immune regulation, we chose DCLK1-expressing mouse colon cancer cells MC38 (C57BL/6J) and CT26 (BALB/c) and used CRISPR-Cas9 technology to establish DCLK1-deficient lines. We implanted 1 × 106 DCLK1wild-type (WT) or DCLK1-/- cells subcutaneously (SC) into the flank of immune-deficient mice or immune-competent mice to investigate the functional interactions between tumor DCLK1 and the immune system. The result showed that in immune-deficient BALB/c nude mice, tumor growth of MC38 DCLK1WT and DCLK1-/- showed no obvious differences, suggesting that DCLK1 has minimal impact on the growth rate of MC38 and CT26 cells in the absence of immune surveillance. On the contrary, in immune-competent mice, MC38 DCLK1-/- tumors showed nominal growth early on followed by gradual clearance, achieving complete regression at the end point, while the MC38 DCLK1WT tumor growth remained normal and unaltered (Figures 1A and B and 2A). The result was repeated in DCLK1-/- CT26 in WT BALB/c mice (Figures 1C and D and 2C). On day 21, we excised and weighed the tumors from DCLK1WT and DCLK1-/- groups, and found a significant difference between the 2 groups (Figure 2B and D). These results indicate that DCLK1 is necessary for tumor growth in the presence of a functioning immune system and plays an important role in tumor immune escape.
      Figure thumbnail gr1
      Figure 1DCLK1 promotes tumor progression through immune escape. (A) Tumor growth of MC38-DCLK1WT and MC38-DCLK1-/- CRC cells implanted in immune-deficient nude mice and immune-competent C57BL/6J mice. (B) Photograph of subcutaneous tumor formation in the BALB/c nude and C57BL/6J mouse model. (C) Tumor growth of CT26-DCLK1WT and CT26-DCLK1-/- CRC cells implanted in immune-deficient nude mice and immune-competent BALB/c mice. (D) Photograph of subcutaneous tumor formation in the BALB/c nude and BALB/c mouse model. (E) CRC MC38-DCLK1WT (top) and MC38-DCLK1-/- (bottom) cells in situ transplantation into the intestine of C57BL/6 mice. Representative photographs of intestinal tumorigenesis (left panel) and anatomic photographs (right panel) as indicated. (F) CRC CT26-DCLK1WT (top) and CT26-DCLK1-/- (bottom) cells in situ transplantation into the intestine of BALB/c mice. Representative photographs of intestinal tumorigenesis (left panel) and anatomic photos (right panel) as indicated. (G) Tumor rechallenge of CRC MC38-DCLK1WT (top) and MC38-DCLK1-/- (middle) cells after prechallenge of MC38-DCLK1-/- cells in C57BL/6J mice. Subcutaneous growth of CRC MC38-DCLK1WT and MC38-DCLK1-/- tumors injected on day 28 without prechallenge (bottom) set as control. (H) Tumor rechallenge of CRC CT26-DCLK1WT (top) and CT26-DCLK1-/- (middle) cells after prechallenge of CT26-DCLK1-/- cells in BALB/c mice. Subcutaneous growth of CRC CT26-DCLK1WT and CT26-DCLK1-/- tumors were injected on day 28 without prechallenge (bottom) as in panel G. Data are presented as means ± SD; n = 6–9 tumors for each group. ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, 2-tailed Student t test.
      Figure thumbnail gr2
      Figure 2Loss of DCLK1 in tumor cells inhibits tumor growth in vivo, related to . (A and B) Deletion of DCLK1 in the MC38 cell line inhibits tumor growth in vivo. (A) MC38-DCLK1WT and single-guide RNA (SgRNA) #2 of MC38-DCLK1-/- cells were injected into C57BL/6J mice. (B) Tumor weight was determined on the 21st day after transplantation and photograph shows subcutaneous MC38 tumor formation in a C57BL/6J mouse model. (C and D) Deletion of DCLK1 in the CT26 cell line inhibits tumor growth in vivo. (C) CT26-DCLK1WT and SgRNA #2 of CT26-DCLK1-/- cells were injected into BALB/c mice. (D) Tumor weight was determined on the 21st day after transplantation and photograph shows subcutaneous CT26 tumor formation in a BALB/c mouse model. Data are represented as means ± SD; n = 6–7 tumors for each group. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, 2-tailed Student t test.
      To simulate in situ tumor growth, we injected DCLK1WT and DCLK1-/- cells into the intestine of C57BL/6J or BALB/c mice. In immunocompetent C57BL/6J mice, the tumor formation ratio was significantly higher in the MC38 DCLK1WT group compared with the DCLK1-/- group, although tumor growth was still small in 2 of the mice in the DCLK1WT group (numbered 4 and 5). Overall, tumors formed in 5 of 9 mice in the DCLK1WT group, but in only 1 of 9 mice in the DCLK1-/- group (Figure 1E). In situ tumor injection in the immunocompetent BALB/c mice showed similar results. That is, we found 5 of 8 mice developed multiple tumors in the CT26 DCLK1WT group, whereas only 1 of 8 mice developed a single occurring tumor in the DCLK1-/- group (Figure 1F), indicating that the DCLK1 molecule is crucial for in situ colon tumor formation.
      Adaptive immunity is characterized by recall responses.
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      We reasoned that if the enhanced rejection of DCLK1-/- cells is indeed a consequence of host adaptive immune activation, their inoculation may be associated with immune memory. To confirm this hypothesis, we implanted C57BL/6J mice and BALB/c mice with syngeneic MC38 DCLK1-/- or CT26 DCLK1-/- cells SC, respectively. On day 21, the DCLK1-/- tumor cells were completely cleared. We rechallenged the mice with DCLK1WT or DCLK1-/- tumor cells on day 28. The result showed that both DCLK1WT and DCLK1-/- cells failed to establish tumors (Figure 1G and H). These results indicate that the lack of DCLK1 promotes a host adaptive immune response with a typical recall response, providing evidence for an immunosuppressive function for DCLK1 in the context of tumorigenesis.

      DCLK1 Ablation Promotes CRC Regression by Modulating MDSC Recruitment and T-Cell Activation

      To further dissect the role of DCLK1 in host immunity, we decided to characterize the role of adaptive immune cells in DCLK1-/- tumor regression. On day 7 after inoculation, we performed flow cytometry analysis of lymphocytes in tumor tissues. Results showed no obvious difference between total CD45+ and CD4+ T cells (Figure 3A and B). In contrast, we found that the infiltration of CD4+CD62L-CD44+ effector T cells increased in MC38 DCLK1-/- tumors (Figure 3C and F), while the infiltration of CD8+ T cells increased significantly both in MC38 and CT26 DCLK1-/- tumors (Figure 3D). Infiltration of CD8+CD62L-CD44+ T cells also increased significantly in MC38 DCLK1-/- tumors (Figure 3E and F). Moreover, the proliferation of CD8+ T cells in the MC38 DCLK1WT tumor was comparatively less robust, as indicated by reduced Ki-67 positivity. No significant difference in the Ki-67 index of CD4+ T cells was detected between DCLK1WT and DCLK1-/- tumors (Figure 3G). In addition, staining of CD8+ T cells in frozen sections from DCLK1WT and DCLK1-/- tumor tissues also showed noticeably increased infiltration in the DCLK1-/- group (Figure 3H).
      Figure thumbnail gr3
      Figure 3DCLK1-depleted tumors show increased T-lymphocyte infiltration. (A) Flow cytometry quantification of CD45+ cell infiltration in CRC DCLK1WT and CRC DCLK1-/- group intratumor tissues. (B) Flow cytometry quantification of total CD4+ T-cell infiltration in CRC DCLK1WT and CRC DCLK1-/- group intratumor tissues. (C) Flow cytometry quantification of effector CD4+ T-cell (CD4+CD44+CD62L-) infiltration in MC38-DCLK1WT and MC38-DCLK1-/- group intratumor tissues. (D) Flow cytometry quantification of CD8+ T-cell infiltration in CRC DCLK1WT and CRC DCLK1-/- group intratumor tissues. (E) Flow cytometry quantification of effector CD8+ T-cell (CD8+CD44+CD62L-) infiltration in MC38-DCLK1WT and MC38-DCLK1-/- group intratumor tissues. (F) Flow cytometry gating strategy applied for identification of T-cell subpopulations. (G) Flow cytometry quantification of Ki-67+ cells as a percentage of infiltrating CD4+ and CD8+ T cells. (H) Frozen sections from MC38-DCLK1WT, MC38-DCLK1-/- and CT26-DCLK1WT, CT26-DCLK1-/- subcutaneous tumor tissues were subjected to immunostaining analysis for CD8a (green) along with 4′,6-diamidino-2-phenylindole (DAPI) for DNA (blue). Data are presented as means ± SD; n = 5–7 tumors for each group. For frozen section staining, 3 sections were stained for each group of tumors. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, 2-tailed Student t test. CD44+CD62L-, effector T cells; CD44+CD62L+, central memory T cells; CD44-CD62L+, naïve T cells; FSC-A, _______; SSC-A, _______.
      To further probe if there were any qualitative differences between CD4+ T cells and CD8+ T cells associated with tumor DCLK1, we analyzed the expression of co-inhibitory surface markers programmed death 1 and cytotoxic T lymphocyte A-4 on T cells. We failed to find any indication of altered T-cell exhaustion marker expression (Figure 4A and B). In addition, we found no difference in NK or NKT cells (Figure 4C and D). Together, these data suggest that differences in infiltrating T cells between DCLK1WT and DCLK1-/- tumor implantations may explain the variable outcomes in DCLK1WT and DCLK1-/- tumor regression.
      Figure thumbnail gr4
      Figure 4DCLK1 deletion boosts antitumor immunity independent of T-cell exhaustion and NK cell infiltration. Flow cytometry quantification of inhibitory marker, including programmed death 1 (PD-1) and cytotoxic T lymphocyte (CTL)A-4, by the percentage of CD8+ and CD4+ tumor-infiltrating T cells in (A) CRC MC38-DCLK1WT, MC38-DCLK1-/- group intratumor tissues, and (B) CRC CT26-DCLK1WT, CT26-DCLK1-/- group intratumor tissues. Flow cytometry quantification of NK cells and NKT cells infiltrating (C) MC38-DCLK1WT, MC38-DCLK1-/- group intratumor tissues and (D) CT26-DCLK1WT, CT26-DCLK1-/- group intratumor tissues. Data are presented as means ± SD; n = 5–7 tumors for each group, 2-tailed Student t test.
      To validate the role of CD4+ and CD8+ T cells in DCLK1-/- CRC tumor regression, we depleted CD4+ or CD8+ T-cell populations by neutralization antibody in mice bearing DCLK1-/- MC38 tumor (Figure 5A and B), and found that removal of either type of T cells led to robust tumor growth exceeding the WT DCLK1 tumor in the control group (Figure 5C). Based on this, we tentatively concluded that the DCLK1WT tumors escape immune surveillance via limiting the infiltration of CD4+ and CD8+ T cells. Subsequently, we assessed the activation status of CD8+ T cells. We found that the interferon-γ and granzyme B–positive T cell percentage of the total CD8+ T cells doubled in MC38 and CT26 DCLK1-/- tumors, suggesting that DCLK1 knockout boosted the activation intensity of individual CD8+ T cells (Figure 6A and B). Together, these findings provide direct functional proof that CD4+ and CD8+ T cells are responsible for the regression of DCLK1-/- tumors.
      Figure thumbnail gr5
      Figure 5CD4+ T and CD8+ T cells are responsible for the clearance of DCLK1-/- tumor cells. (A) Flow cytometry analysis of antibody-mediated T-cell depletion. T-cell populations of spleen, lymph node, and thymus tissue from mice commencing 8-day neutralizing antibody treatment before tumor inoculation. (B) Flow cytometry examination of T-cell populations of spleen and thymus tissue from tumor-bearing animals with continuing T-cell depletion antibody treatment. (C) Tumor growth kinetics after transplantation of CRC MC38-DCLK1WT or MC38-DCLK1-/- cells into C57BL/6J mice receiving anti-CD4 or anti-CD8a neutralizing antibodies. n = 6–8 tumors for each group, 2-tailed Student t test, ∗∗∗∗P < .0001. Ctrl, control; PE-A, _____.
      Figure thumbnail gr6
      Figure 6DCLK1-expressing tumors show a higher proportion of MDSC infiltration, which inhibits T-cell proliferation. (A) Flow cytometry quantification of CD8+IFN-γ+ cell and CD8+GranzymeB+ cell percentage in CRC DCLK1WT and DCLK1-/- subcutaneous tumors from immunocompetent mice (top panel: MC38-DCLK1WT, MC38-DCLK1-/- groups; bottom panel: CT26-DCLK1WT, CT26-DCLK1-/- groups). (B) Flow cytometry gating strategy for IFN-γ and GranzymeB staining of CD8+ T cells applied for identification of the CD8+ T-cell activation status. (C) Representative flow cytometry quantification of the MDSC subpopulation in MC38-DCLK1WT and MC38-DCLK1-/- group intratumor tissues (left panel), and CT26-DCLK1WT and CT26-DCLK1-/- group intratumor tissues (right panel). (D) Flow cytometry gating strategy applied for identification of the MDSC subpopulation. Ly6G+ subpopulation, G-MDCS; Ly6C+ subpopulation, monocytic MDSC (M-MDSC). (E) Representative flow cytometry quantification of arginase 1 (ARG-1) (left) and nitric oxide synthase 2 (NOS2) (right) percentage of MDSCs in MC38-DCLK1WT and MC38-DCLK1-/- group intratumor tissues (upper panel), and CT26-DCLK1WT and CT26-DCLK1-/- group intratumor tissues (lower panel). (F) Flow cytometry results of the CFSE-labeled T-cell proliferation. CD8+T cells, activated by anti-CD3/28 (4 μg/mL), were co-cultured with MDSCs sorted from the subcutaneous MC38 DCLK1WT and DCLK1-/- tumors. The proliferation of CD8+ T cells is indicated by percentage. Data are presented as means ± SD; n = 5–7 tumors for each group. ∗P < .05, ∗∗P < .01, and ∗∗∗∗P < .0001, 2-tailed Student t test. FMO, _______; FSC-H, ______; IFN, interferon; SSC-A, _____.
      Mounting evidence has suggested that the accumulation of MDSCs in the tumor microenvironment can inhibit the infiltration and function of T cells and promote the immune escape of tumors.
      • Gabrilovich D.I.
      • Nagaraj S.
      Myeloid-derived suppressor cells as regulators of the immune system.
      ,
      • Di Mitri D.
      • Toso A.
      • Alimonti A.
      Molecular pathways: targeting tumor-infiltrating myeloid-derived suppressor cells for cancer therapy.
      • Veglia F.
      • Perego M.
      • Gabrilovich D.
      Myeloid-derived suppressor cells coming of age.
      • Gabrilovich D.I.
      Myeloid-derived suppressor cells.
      Next, we analyzed immunosuppressive cell infiltration in DCLK1WT and DCLK1-/- tumors. As shown by the flow cytometry results, there was a significant decrease in G-MDSC infiltration in the DCLK1-/- group (Figure 6C and D). Subsequently, to clarify whether DCLK1 regulates the MDSC function besides recruitment, we detected the expression of arginase-1 and nitric oxide synthase 2, which suppress T-cell function.
      • Veglia F.
      • Perego M.
      • Gabrilovich D.
      Myeloid-derived suppressor cells coming of age.
      ,
      • Gabrilovich D.I.
      Myeloid-derived suppressor cells.
      As shown by the results, these 2 immunosuppressive molecules show similar intensity between DCLK1WT and DCLK1-/- groups (Figure 6E). Furthermore, to determine whether MDSCs from these 2 groups impede T-cell proliferation, we isolated the mouse spleen CD8+ T cells, labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3 and anti-CD28 antibody in the presence of equal numbers of MDSCs sorted from mouse subcutaneous MC38 DCLK1WT and DCLK1-/- tumors. MDSCs from DCLK1WT and DCLK1-/- groups showed similar inhibition of T-cell proliferation in vitro (Figure 6F). However, because MDSC infiltration in the DCLK1-/- CT26 tumor is severely defective, we did not acquire enough MDSCs from CT26 tumors. These results indicate that MDSCs from DCLK1WT tumors do not possess more potent T-cell inhibition than DCLK1-/- tumors. In brief, these results implicate positive regulation of G-MDSC recruitment in DCLK1-expressing tumors, but no alteration on the T-cell inhibitory capacity of MDSCs.

      MDSC-Recruiting Chemokines, CXCL1 and CXCL2, Are Highly Expressed in DCLK1WT CRC Tumor Cells

      To explore the mechanism of increased intratumoral MDSCs in DCLK1-expressing tumors, we performed the RNA sequencing (RNA-seq) analysis of MC38 DCLK1WT and DCLK1-/- cells. KEGG and Gene Ontology (GO) enrichment analysis identified remarkable differences in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway between DCLK1WT and DCLK1-/- tumors (Figure 7A and B). Correlation analysis of the RNA-seq results showed that genes in the ERK pathway are correlated with DCLK1 (Figure 7C). Next, we confirmed that DCLK1 expression is correlated positively to ERK pathway activation in human CRC through analysis of the colorectal adenocarcinoma (COAD) The Cancer Genome Atlas (TCGA) cohort (Figure 7D). According to recent studies, CXCL1 and CXCL2 are both ligands of CXCR2 and play an important role in G-MDSC recruitment.
      • Katoh H.
      • Wang D.
      • Daikoku T.
      • Sun H.
      • Dey S.K.
      • Dubois R.N.
      CXCR2-expressing myeloid-derived suppressor cells are essential to promote colitis-associated tumorigenesis.
      ,
      • Wang D.
      • Sun H.
      • Wei J.
      • Cen B.
      • DuBois R.N.
      CXCL1 is critical for premetastatic niche formation and metastasis in colorectal cancer.
      Notably, CXCL1 and CXCL2 dramatically decreased in the DCLK1-/- group (Figure 7E). Furthermore, STRING-based protein–protein interaction analysis suggested a relationship between DCLK1 and CXCL1 and CXCL2 through the ERK pathway (Figure 7F).
      Figure thumbnail gr7
      Figure 7The recruitment factors for MDSCs (CXCL1 and CXCL2) are highly expressed in DCLK1-high colon carcinoma. (A) KEGG analysis shows that genes affected by DCLK1 knockout are enriched in the MAPK–ERK pathway. (B) GO enrichment analysis suggests that down-regulated genes are enriched in processes related to Erk1 and Erk2 cascade. (A and B) Red bars indicate up-regulated genes, while blue bars indicate down-regulated genes. The numbers around the bars are the number of genes in corresponding categories. (C) Expression correlations between DCLK1 and genes in the MAPK–ERK pathway. The X-axis shows Pearson correlation coefficients, and the Y-axis shows –log10 (P values) for the correlations. (D) Co-expression relationships between DCLK1 and the MAPK–ERK pathway in TCGA-COAD project (n = 480). (E) Expression differences among genes in the MAPK–ERK pathway. Levels are normalized by row. (F) Co-expression relationships identified in this study suggest potential crosstalk between DCLK1 and the MAPK–ERK pathway via CXCL1/CXCL2. Nodes represent proteins, and edges represent protein–protein associations, whose colors represent levels of evidence. Relative messenger RNA expression levels of CXCL1 and CXCL2 in (G) MC38-DCLK1WT, MC38-DCLK1-/-, and (I) CT26-DCLK1WT, CT26-DCLK1-/- tumor cells and (K) human CRC cell lines SW480-DCLK1WT, SW480-DCLK1-/-, HCT116-DCLK1WT, and (M) HCT116 DCLK1OE by RT-PCR analysis. ELISA results of cell supernatants of (H and J) CRC cell lines MC38-DCLK1WT, MC38-DCLK1-/-, CT26-DCLK1WT, and CT26-DCLK1-/-, and (L and N) human CRC cell line SW480-DCLK1WT, SW480-DCLK1-/-, HCT116-DCLK1WT, and HCT116 DCLK1OE. (O) High expression of DCLK1 promotes MDSC recruitment in vitro. MC38 or CT26 cells were inoculated subcutaneously into WT C57BL/6J or BALB/c mice. On the seventh day, the tumor tissues were stripped and tumor-infiltrating MDSCs were isolated. MDSCs (2 × 105) were placed into the Transwell upper chamber. DCLK1WT and DCLK1-/- cell-conditioned medium were added into the lower chamber of the Transwell, and the migration of MDSCs was observed 48 hours later. Data are presented as means ± SD. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, 2-tailed Student t test. Akt, ______; ECM, ______; EGFR, ______; NOD, ______; PI3K, ______.
      To verify the results of the transcriptome analysis, we examined the expression of CXCR2 ligands by reverse-transcription polymerase chain reaction (RT-PCR). CXCL1 and CXCL2 expression were markedly higher in MC38 or CT26 DCLK1WT cells compared with DCLK1-/- cells (Figure 7G and I). To further confirm these results in human CRC lines, we selected SW480 with high expression of DCLK1, and HCT116 with low expression of DCLK1. After knocking out or overexpressing DCLK1 in these 2 CRC lines, respectively, we tested the expression of CXCL1 and CXCL2 and found decreased expression of both in SW480 DCLK1-/- CRC lines (Figure 7K). Conversely, when we forced the expression of DCLK1 in HCT116 cells, the expression of CXCL1 and CXCL2 increased (Figure 7M). We further confirmed the CXCL1 and CXCL2 expression in cell culture supernatant using enzyme-linked immunosorbent assay (ELISA). Protein levels of CXCL1 and CXCL2 in DCLK1-/- medium were lower than those in DCLK1WT medium, whereas DCLK1 overexpression led to higher levels (Figure 7H, J, L, and N). These data together show that the expression of chemokines CXCL1 and CXCL2, which are known to recruit MDSCs, are regulated by DCLK1. To prove that DCLK1 regulates MDSC recruitment by promoting the expression of CXCL1 and CXCL2, we extracted MC38 and CT26 tumor tissues inoculated SC in mice, isolated MDSCs, and cultured them in a Transwell upper chamber. The conditioned medium of DCLK1WT and DCLK1-/- tumor cells were added to the lower chamber and the migratory ability of MDSCs was detected. MDSC migration in the DCLK1WT conditioned medium was increased significantly compared with the DCLK1-/- group (Figure 7O). These data show that DCLK1 promotes the expression of chemokines CXCL1 and CXCL2, which are known to recruit MDSCs in the tumor microenvironment.

      DCLK1 Induces CXCL1/CXCL2 Expression via the ERK Pathway

      The earlier-described results indicate that the expression of CXCL1 and CXCL2 is regulated by the expression of DCLK1. Because we found remarkable differences in ERK pathway enrichment in the DCLK1WT group compared with the DCLK1-/- cell lines, we sought to assess this finding at the protein level. Western blot showed significantly decreased ERK phosphorylation in DCLK1-/- tumor cells (Figure 8A). To further investigate whether DCLK1 regulates CXCL1 and CXCL2 through the ERK pathway, we used ERK-specific inhibitor SCH772984
      • Chaikuad A.
      • Tacconi E.M.
      • Zimmer J.
      • Liang Y.
      • Gray N.S.
      • Tarsounas M.
      • Knapp S.
      A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics.
      to treat MC38, CT26, SW480, and DCLK1-overexpressing HCT116 cells, and detected the expression changes of CXCL1 and CXCL2 by RT-PCR. The expression of CXCL1 and CXCL2 noticeably decreased after inhibition of the ERK pathway by SCH772984 (Figure 8B). According to the EPD eukaryotic promoter database (https://epd.epfl.ch//index.php) generated from validated experimental results, we found that c-Myc, a key downstream transcription factor of the ERK pathway,
      • Marampon F.
      • Ciccarelli C.
      • Zani B.M.
      Down-regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non muscle-derived human tumors.
      ,
      • Tsai W.B.
      • Aiba I.
      • Long Y.
      • Lin H.K.
      • Feun L.
      • Savaraj N.
      • Kuo M.T.
      Activation of Ras/PI3K/ERK pathway induces c-Myc stabilization to upregulate argininosuccinate synthetase, leading to arginine deiminase resistance in melanoma cells.
      has specific binding sites on the promoter of CXCL1 and CXCL2. Next, we detected the expression of c-Myc in the nucleus. The results showed that there was a positive correlation between the expression of c-Myc and DCLK1 in the nucleus (Figure 8C).
      Figure thumbnail gr8
      Figure 8DCLK1 regulates MDSC recruitment factor CXCL1 through the ERK pathway. (A) Immunoblot results of ERK protein phosphorylation in DCLK1-knockout MC38, CT26, and SW480 cells compared with control cells, and DCLK1-overexpressing HCT116 cells compared with control cells. (B) RT-PCR results for CXCL1 and CXCL2 in DCLK1WT, DCLK1-/- cells and DCLK1WT, DCLK1OE treated with or without SCH772984 (ERK inhibitor) (1 μmol/L, 48 hours). (C) Western blot results for nuclear protein extracts that have validated binding to the CXCL1 and CXCL2 promoter region. (D) Tumor growth in mice injected SC with DCLK1WT, DCLK1-/- cells, and CXCL1 or CXCL2 rescued DCLK1-/- cells, the tumor-growth kinetics were measured every 4–5 days. (E) Flow cytometry quantification of CD8+ T cells and the MDSC percentage for subcutaneous MC38-DCLK1WT, MC38-DCLK1-/-, and CXCL1 rescued MC38-DCLK1-/- tumors on the seventh day after implantation. (F) Four cell lines, MC38 DCLK1WT, MC38 DCLK1-/-, MC38 DCLK1-/--CXCL1OE, and MC38 DCLK1-/--CXCL2OE were inoculated with 1.5 × 106 cells per mouse in situ in the intestine, and the tumor growth of each group was compared after the mice were killed on the 21st day after tumor inoculation. (G) Subcutaneous tumor growth in mice injected with MC38-DCLK1WT and DCLK1-/- cells treated daily with SB265610 (CXCR2 inhibitor, 2 mg/kg body weight) or PBS control starting from tumor inoculation. (H) Flow cytometry quantification of CD8+T cells and the MDSC percentage for subcutaneous tumors receiving treatment on the seventh day after implantation. (I) Mice were inoculated with MC38 DCLK1-WT and DCLK1-knockout (KO) colon cancer cell lines in situ and given CXCR2-receptor inhibitor SB265610 intraperitoneally for 14 days after 7 days of inoculation. After day 21, mice were killed and intestinal dissection was performed to compare tumor formation between the inhibitor-dosed and nondosed groups. Data are presented as means ± SD; n = 5–7 tumors for each group. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, 2-tailed Student t test. DMSO, dimethyl sulfoxide; ERKi, _______; mRNA, messenger RNA; p-ERK, _______.
      To further explore the function of CXCL1 and CXCL2 in DCLK1+ tumors, we rescued the expression of CXCL1 and CXCL2 in the DCLK1-/- MC38 cell line and transplanted the DCLK1WT, DCLK1-/-, DCLK1-/--CXCL1OE, and DCLK1-/--CXCL2OE cell lines into the flanks of C57BL/6J mice. CXCL1 rescue led to a tumor growth rate for DCLK1-/- CRC cells similar to WT cells. However, tumor growth was not restored by CXCL2 overexpression (Figure 8D). Notably, the rescue of CXCL1 also reduced the infiltration of CD8+ T cells and enhanced the infiltration of MDSCs (Figure 8E). To validate this, we established an in situ mouse model of intestinal tumor and found that MC38 DCLK1WT cells had the highest rate of tumorigenesis, including 2 of 6 mice showing a heavy tumor burden (tumor size, >5 mm), 2 mice with moderate burden (tumor size, 2–4 mm), and 1 mouse with mild tumor burden (tumor size, 1–2 mm). In contrast, only 2 mice in the DCLK1-/- group had visible tumors, and the tumor burden was relatively low (tumor size, 1–2 mm). As expected, CXCL1 (but not CXCL2) overexpression in DCLK1-/- tumors led to exceeding in situ tumor growth and 5 of 6 mice developed multiple tumors, the tumors of mice 1–3 were more than 5 mm in diameter, and mice 4 and 5 were approximately 3 mm in diameter, which was consistent with the subcutaneous tumor transplantation assays. In agreement with the subcutaneous tumorigenesis assay, DCLK1-/--CXCL2OE inoculation had no such effect, indicating the dominant role of CXCL1 in DCLK1-mediated tumorigenesis (Figure 8F).
      CXCR2 has been implicated in the progression of a wide range of tumor types.
      • Yang G.
      • Rosen D.G.
      • Liu G.
      • Yang F.
      • Guo X.
      • Xiao X.
      • Xue F.
      • Mercado-Uribe I.
      • Huang J.
      • Lin S.H.
      • Mills G.B.
      • Liu J.
      CXCR2 promotes ovarian cancer growth through dysregulated cell cycle, diminished apoptosis, and enhanced angiogenesis.
      ,
      • Singh S.
      • Varney M.
      • Singh R.K.
      Host CXCR2-dependent regulation of melanoma growth, angiogenesis, and experimental lung metastasis.
      Recent research reported that antagonists targeting CXCR2 showed efficacy in inflammatory response and against colorectal tumors.
      • Ning Y.
      • Labonte M.J.
      • Zhang W.
      • Bohanes P.O.
      • Gerger A.
      • Yang D.
      • Benhaim L.
      • Paez D.
      • Rosenberg D.O.
      • Nagulapalli Venkata K.C.
      • Louie S.G.
      • Petasis N.A.
      • Ladner R.D.
      • Lenz H.J.
      The CXCR2 antagonist, SCH-527123, shows antitumor activity and sensitizes cells to oxaliplatin in preclinical colon cancer models.
      To examine whether modulation of CXCR2 alters MDSC recruitment to the tumor microenvironment, we administered the CXCR2 antagonist (SB265610) to DCLK1WT MC38 tumor-bearing mice. It showed that administration of CXCR2 antagonist efficiently inhibited tumorigenesis of DCLK1WT cells (Figure 8G), along with promoted infiltration of CD8+ T cells; the infiltration of MDSCs, however, was suppressed accordingly (Figure 8H). We investigated the antitumor effect of SB265610 with the in situ model of DCLK1 cells. We found that intestinal tumorigenesis in DCLK1WT cell transplanted mice remained severe, 3 mice showed 3- to 4-mm diameter tumor formation, and the other 3 mice showed 2- to 3-mm tumors, including 2 mice with multiple tumor formation. Tumor burden was milder in mice treated with SB265610, only 4 of the 6 mice had tumor formation with a diameter of 1–3 mm, and all developed individually. Because of the tumor-suppressive effect of DCLK1-knockout in situ, accurately assessing the inhibitory effect of SB265610 is not possible. However, in the DCLK1-knockout cell inoculated group, only 1 of 6 mice treated with SB265610 developed a tumor with a diameter of 1 mm, while 2 of 6 mice treated with vehicle developed tumors that were 2 mm in diameter (Figure 8I). Collectively, these findings suggest that CXCL1–CXCR2 interaction downstream of DCLK1 plays a critical role in the tumorigenesis and progression of DCLK1+ CRC.

      Clinical Expression of DCLK1 and CXCL1 in CRC Patients and the Relevance of DCLK1 and MDSC Signature

      Our findings indicate that DCLK1 can affect the tumor immune microenvironment by recruiting MDSCs and promoting tumor immune escape. To verify this in clinical samples, we analyzed the TCGA COAD cohort with an established MDSC gene signature,
      • Liao W.
      • Overman M.J.
      • Boutin A.T.
      • Shang X.
      • Zhao D.
      • Dey P.
      • Li J.
      • Wang G.
      • Lan Z.
      • Li J.
      • Tang M.
      • Jiang S.
      • Ma X.
      • Chen P.
      • Katkhuda R.
      • Korphaisarn K.
      • Chakravarti D.
      • Chang A.
      • Spring D.J.
      • Chang Q.
      • Zhang J.
      • Maru D.M.
      • Maeda D.Y.
      • Zebala J.A.
      • Kopetz S.
      • Wang Y.A.
      • DePinho R.A.
      KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer.
      and identified a strong positive correlation with DCLK1 expression (Figure 9A). Notably, CD33, a classic marker of human MDSCs, showed a significant correlation with DCLK1 (Figure 9B). Using polychromatic fluorescent staining, we evaluated the expression of DCLK1, CXCL1, and CD33 in primary CRC and paracancerous tissues. The results showed that high expression of DCLK1 was accompanied by high expression of CD33 and an increased level of CXCL1 in colon cancer tissues compared with the DCLK1-low tumor tissues and adjacent tissues (Figure 9C).
      Figure thumbnail gr9
      Figure 9The expression of DCLK1 is associated with CXCL1 expression, MDSC infiltration, and poor prognosis in colon cancer patients. (A) Expression correlations between DCLK1 and known genes that actively are involved in MDSC regulation. The transcriptome profiling samples from patients with colon cancer (TCGA-COAD project) were used. (B) The correlation scatterplot shows a significant correlation between the expression of DCLK1 and CD33. The same batch of profiling samples as in panel A was used. Expression levels were log(ln)-transformed. (C) Fluorescence staining of CRC tissues shows that the expression and release of CD33 and CXCL1 in tumor tissues with high expression of DCLK1 are significantly higher than those in adjacent and low DCLK1 expression tissues. (D) Expression levels of DCLK1 have a notable impact on disease progression-free survival in patients with stage I/II. (E) Expression levels of DCLK1 have a notable impact on overall survival of stage I patients.
      We further clarified the prognostic value of DCLK1 in CRC patients of the TCGA COAD project and found that high DCLK1 expression significantly correlated to poor prognosis for stage I/II CRC patients. We speculate that the main reason for this may be DCLK1-mediated immunosuppression and the resulting effect on tumor recurrence and metastasis (Figure 9D). In addition, stage I patients with high expression of DCLK1 have a worse prognosis for overall survival than those with lower expression, and the hazard ratio was 8.081, suggesting that DCLK1 is a predictor for the overall survival of patients with stage I CRC, but high DCLK1 expression is not a good predictor of overall patient survival for the rest of the stages (Figure 9E). This finding suggests that the detection of DCLK1 expression can predict the survival of patients with early CRC, which may be of significance for clinical diagnosis and treatment.

      Discussion

      DCLK1 is well known as a molecule that is highly expressed in CRC,
      • Chandrakesan P.
      • Yao J.
      • Qu D.
      • May R.
      • Weygant N.
      • Ge Y.
      • Ali N.
      • Sureban S.M.
      • Gude M.
      • Vega K.
      • Bannerman-Menson E.
      • Xia L.
      • Bronze M.
      • An G.
      • Houchen C.W.
      Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells.
      ,
      • Liu W.
      • Wang S.
      • Sun Q.
      • Yang Z.
      • Liu M.
      • Tang H.
      DCLK1 promotes epithelial-mesenchymal transition via the PI3K/Akt/NF-kappaB pathway in colorectal cancer.
      ,
      • Gao T.
      • Wang M.
      • Xu L.
      • Wen T.
      • Liu J.
      • An G.
      DCLK1 is up-regulated and associated with metastasis and prognosis in colorectal cancer.
      pancreatic cancer,
      • Westphalen C.B.
      • Takemoto Y.
      • Tanaka T.
      • Macchini M.
      • Jiang Z.
      • Renz B.W.
      • Chen X.
      • Ormanns S.
      • Nagar K.
      • Tailor Y.
      • May R.
      • Cho Y.
      • Asfaha S.
      • Worthley D.L.
      • Hayakawa Y.
      • Urbanska A.M.
      • Quante M.
      • Reichert M.
      • Broyde J.
      • Subramaniam P.S.
      • Remotti H.
      • Su G.H.
      • Rustgi A.K.
      • Friedman R.A.
      • Honig B.
      • Califano A.
      • Houchen C.W.
      • Olive K.P.
      • Wang T.C.
      Dclk1 defines quiescent pancreatic progenitors that promote injury-induced regeneration and tumorigenesis.
      ,
      • Qu D.
      • Weygant N.
      • Yao J.
      • Chandrakesan P.
      • Berry W.L.
      • May R.
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      • Li M.
      • Wang T.C.
      • An G.
      • Clendenin C.
      • Stanger B.Z.
      • Houchen C.W.
      Overexpression of DCLK1-AL increases tumor cell invasion, drug resistance, and KRAS activation and can be targeted to inhibit tumorigenesis in pancreatic cancer.
      renal cancer, and other tumors.
      • Ge Y.
      • Weygant N.
      • Qu D.
      • May R.
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      • Drake M.
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      • Bronze M.S.
      • Tomasek J.J.
      • An G.
      • Houchen C.W.
      Alternative splice variants of DCLK1 mark cancer stem cells, promote self-renewal and drug-resistance, and can be targeted to inhibit tumorigenesis in kidney cancer.
      ,
      • Weygant N.
      • Qu D.
      • May R.
      • Tierney R.M.
      • Berry W.L.
      • Zhao L.
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      • Chandrakesan P.
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      • Murphy N.T.
      • Li J.D.
      • Sureban S.M.
      • Schlosser M.J.
      • Tomasek J.J.
      • Houchen C.W.
      DCLK1 is a broadly dysregulated target against epithelial-mesenchymal transition, focal adhesion, and stemness in clear cell renal carcinoma.
      In various models and human samples, it regulates the development, progression, metastasis, and epithelial–mesenchymal transition of tumors. Studies have shown that inhibition of DCLK1 expression can slow down tumor progression and metastasis.
      • Weygant N.
      • Qu D.
      • Berry W.L.
      • May R.
      • Chandrakesan P.
      • Owen D.B.
      • Sureban S.M.
      • Ali N.
      • Janknecht R.
      • Houchen C.W.
      Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1.
      ,
      • Ge Y.
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      • Qu D.
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      • Zhao K.L.
      • Drake M.
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      • Bronze M.S.
      • Tomasek J.J.
      • An G.
      • Houchen C.W.
      Alternative splice variants of DCLK1 mark cancer stem cells, promote self-renewal and drug-resistance, and can be targeted to inhibit tumorigenesis in kidney cancer.
      ,
      • Sureban S.M.
      • May R.
      • Weygant N.
      • Qu D.
      • Chandrakesan P.
      • Bannerman-Menson E.
      • Ali N.
      • Pantazis P.
      • Westphalen C.B.
      • Wang T.C.
      • Houchen C.W.
      XMD8-92 inhibits pancreatic tumor xenograft growth via a DCLK1-dependent mechanism.
      We observed similar results consistent with previous studies, such as DCLK1 knockout impeded the tumor cell migration in MC38 and CT26 tumor cell lines (Figure 10A and B). However, some of our findings differed from previous studies. For example, we found no change in cell proliferation between DCLK1WT and DCLK1-/- groups of mouse CRC cell lines (Figure 10C–F). Accordingly, there was no significant difference in in vivo tumor growth in immune-deficient mice between the 2 groups (Figure 1A and C), which differed from previous results in human CRC cell lines.
      • Makino S.
      • Takahashi H.
      • Okuzaki D.
      • Miyoshi N.
      • Haraguchi N.
      • Hata T.
      • Matsuda C.
      • Yamamoto H.
      • Mizushima T.
      • Mori M.
      • Doki Y.
      DCLK1 integrates induction of TRIB3, EMT, drug resistance and poor prognosis in colorectal cancer.
      We also explored cell-cycle changes in DCLK1WT and DCLK1-/- cell lines and found no alteration (Figure 10G and H). This observation is critical for our collective research on this molecule because the former would attribute the role of DCLK1 in cell-intrinsic regulation while our finding put this molecule front and center in host immune regulation.
      Figure thumbnail gr10
      Figure 10DCLK1 deletion reduces the migration ability of tumor cells, but not proliferation, in vitro. (A and B) The cell migration ability of DCLK1WT and DCLK1-/- cells was analyzed by Transwell assays. Significantly reduced cell migration was detected after down-regulation of DCLK1 in both MC38 and CT26 cells. (C–F) The deletion of DCLK1 did not affect tumor proliferation of MC38 and CT26 cell lines in vitro. (C and E) Representative images of the colony-formation assay are shown. (D and F) The Cell Counting Kit-8 (CCK-8) experiments show the growth curve of the MC38 and CT26 cell lines. (G and H) The cell cycle of MC38-DCLK1WT, MC38-DCLK1-/- and CT26-DCLK1WT, CT26-DCLK1-/- were analyzed by flow cytometry. Right: Representative images of cell-cycle assays and the percentage of each phase is shown. Data are presented as means ± SD, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, 2-tailed Student t test.
      In this study, we discovered that DCLK1 can lead to immunosuppression in CRC and promote tumor progression. By constructing in situ and subcutaneous tumor models of mouse intestinal cancer, comparing tumor formation in immunocompetent and immunodeficient mice, and combining differential analysis of tumor-infiltrating immune cells, we confirm the regulatory role of DCLK1 on inhibitory tumor microenvironment in vivo. We note that current studies on DCLK1-mediated tumor immune escape are based mainly on transcriptional or bioinformatic analysis and cell-level verification,
      • Wu X.
      • Qu D.
      • Weygant N.
      • Peng J.
      • Houchen C.W.
      Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments.
      ,
      • Chandrakesan P.
      • Panneerselvam J.
      • May R.
      • Weygant N.
      • Qu D.
      • Berry W.R.
      • Pitts K.
      • Stanger B.Z.
      • Rao C.V.
      • Bronze M.S.
      • Houchen C.W.
      DCLK1-isoform2 alternative splice variant promotes pancreatic tumor immunosuppressive M2-macrophage polarization.
      but in vivo studies have not been performed to date.
      Immune escape plays an important role in tumor occurrence and development. A suspected role for DCLK1 in this process has been inferred from numerous reports.
      • Wu X.
      • Qu D.
      • Weygant N.
      • Peng J.
      • Houchen C.W.
      Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments.
      ,
      • Cao Z.
      • Weygant N.
      • Chandrakesan P.
      • Houchen C.W.
      • Peng J.
      • Qu D.
      Tuft and cancer stem cell marker DCLK1: a new target to enhance anti-tumor immunity in the tumor microenvironment.
      However, mechanistic insights remain elusive. In support of the “immune-centric” role of DCLK1, our team recently found that DCLK1 could promote the expression of programmed cell death-ligand 1 on pancreatic tumor cells via up-regulating the YAP molecule. YAP is a potent regulator of the Hippo pathway that is an integral part of host immunity.
      • Yan R.
      • Li J.
      • Zhou Y.
      • Yao L.
      • Sun R.
      • Xu Y.
      • Ge Y.
      • An G.
      Inhibition of DCLK1 down-regulates PD-L1 expression through Hippo pathway in human pancreatic cancer.
      Recent studies also have shown that DCLK1 is correlated with a variety of immune cells, including CD8+ T cells and CD4+ T cells in the CRC immune microenvironment.
      • Wu X.
      • Qu D.
      • Weygant N.
      • Peng J.
      • Houchen C.W.
      Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments.
      In our study, in addition to the close relationship between DCLK1, CD4+ T cells, and CD8+ T cells, we also found that DCLK1 plays a crucial role in the recruitment of immunosuppressive MDSCs, a key group of cells that have not been experimentally linked to DCLK1. MDSCs, a heterogeneous population of immature myeloid immune cells, have been reported to be indispensable for tumor angiogenesis, invasion, and metastasis.
      • Kumar V.
      • Patel S.
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      The nature of myeloid-derived suppressor cells in the tumor microenvironment.
      ,
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      • Gabrilovich D.
      Myeloid-derived suppressor cells coming of age.
      ,
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      • Gabrilovich D.I.
      History of myeloid-derived suppressor cells.
      The CXCL–CXCR2 axis is required in the recruitment and trafficking of MDSCs. CXCL1, CXCL2, CXCL3, and CXCL5 are all important ligands of CXCR2 and contribute to the infiltration of MDSCs into the colonic mucosa and tumors.
      • Katoh H.
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      CXCR2-expressing myeloid-derived suppressor cells are essential to promote colitis-associated tumorigenesis.
      ,
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      • Biankin A.V.
      • Nibbs R.J.B.
      • Barry S.T.
      • Sansom O.J.
      • Morton J.P.
      CXCR2 inhibition profoundly suppresses metastases and augments immunotherapy in pancreatic ductal adenocarcinoma.
      In the TME, the accumulation of MDSC cells is one of the most important mechanisms of tumor immune escape, and targeting MDSCs is a promising therapeutic avenue.
      • Wesolowski R.
      • Markowitz J.
      • Carson 3rd, W.E.
      Myeloid derived suppressor cells - a new therapeutic target in the treatment of cancer.
      ,
      • Yang L.
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      • Fukuda K.
      • Fingleton B.
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      • Shyr Y.
      • Matrisian L.M.
      • Carbone D.P.
      • Lin P.C.
      Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis.
      As for the mechanism of MDSC recruitment by DCLK1, our study found that activation of the MAPK/ERK pathway facilitates CXCL1 expression, and the c-Myc transcription factor downstream of ERK was found to have binding sites with the CXCL1 promoter, but whether additional pathways involved in the DCLK1-mediated regulation of CXCL1/2 remains to be determined subsequent studies. Therefore, the present findings are a vital supplement to the current studies, without which the reality of this phenomenon in vivo would remain unknown.
      Immune checkpoint blockade (ICB) therapy is a major breakthrough in cancer treatment. However, at present, ICB is only highly applicable to non–small cell lung cancer, melanoma, and renal cancers. For patients with CRC, ICB shows promise only in the small subset of patients with damaged mismatch repair or microsatellite instability-high tumors.
      • Le D.T.
      • Uram J.N.
      • Wang H.
      • Bartlett B.R.
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      • Anders R.A.
      • Eshleman J.R.
      • Vogelstein B.
      • Diaz Jr., L.A.
      PD-1 blockade in tumors with mismatch-repair deficiency.
      In this study, we explored the role of DCLK1 with both microsatellite instability MC38 and MSS CT26 tumor cell lines and reached the same conclusion. Therefore, our study can be viewed as a mismatch repair–independent approach. In addition, many reports have indicated that CRC patients remain resistant to ICB. The possible mechanisms include the decrease in T-cell infiltration, the absence of tumor-specific antigens, and the increase in immunosuppressive cell infiltration.
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer-immune set point.
      ,
      • Sharma P.
      • Hu-Lieskovan S.
      • Wargo J.A.
      • Ribas A.
      Primary, adaptive, and acquired resistance to cancer immunotherapy.
      Recently, attention is being paid to MDSCs, which may be partly responsible for the failure of ICB treatment.
      • Davis R.J.
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      Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid-derived suppressor cells with a selective inhibitor of PI3Kdelta/gamma.
      ,
      • Weber R.
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      • Utikal J.
      • Umansky V.
      Myeloid-derived suppressor cells hinder the anti-cancer activity of immune checkpoint inhibitors.
      The combination of MDSC-targeted drugs and ICB also has been performed in a number of clinical trials, such as NCT04599140. In this Clinical Trial, the efficacy of nivolumab in combination with the CXCR1/2-receptor antagonist SX-682 was evaluated in patients with MSS colon cancer, but the results have not yet been shown.
      Collectively, we found that DCLK1 promotes the immunosuppressive TME through recruitment of MDSCs, and targeting DCLK1 may be a promising target for immunotherapy in CRC. Our work sheds light on how DCLK1 contributes to CRC immune evasion by allowing tumor cells to escape from immunosurveillance. Furthermore, our results provide a rationale to develop CXCR2 antagonists and DCLK1 inhibitors as therapeutic approaches to subverting tumor-induced immunosuppression. For early colon cancer patients with high DCLK1 expression, we may be able to combine DCLK1 inhibitors (such as LRRK2-IN-1, XMD8-92, and DCLK1-IN-1) or CXCR2 inhibitors (navarixin) with existing antineoplastic treatment to overcome immune tolerance and prolong progression-free survival. In addition, DCLK1 may serve as a biomarker to predict the survival of patients and provide guidance for clinical treatment.

      Materials and Methods

      Cell Lines

      The MC38, CT26, SW480, and HCT116 CRC cell lines and 293FT cells were obtained from American Type Culture Collection (Manassas, VA). MC38 and CT26 cells were cultured with RPMI1640 (Gibco), SW480 cells were cultured in L-15 medium, HCT116 cells were cultured in McCoy's 5A medium, and 293FT cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Gibco). All medium was supplemented with fetal bovine serum (FBS) (10%; Ausbian, Australia), penicillin (100 U/mL), and streptomycin (100 mg/mL). The incubator maintained an atmosphere of 5% CO2 at 37ºC for the MC38, CT26, HCT116, and 293FT cell lines. For the SW480 cell line, CO2-free culture conditions were used as recommended by American Type Culture Collection.

      Gene Deletion of DCLK1 by the CRISPR/Cas9 System

      DCLK1-deficient cells were constructed through CRISPR/Cas9 technology. According to the principle of the CRISPR/Cas9 system, we designed the single-guide RNA to target the DCLK1 gene. The sequences used for mouse DCLK1 are as follows: 5’-GCATTTTGATGAGCGGGACA-3’ and 5’-AGCAGGGGGTCCCGTGTGAA-3’, and for human DCLK1 is 5’-CACCGGAGTAGAGAGCTGACTACCA-3’. The single-guide RNA sequences were ligated with linearized LentiCRISPR V2 plasmids. Two plasmids, psPAX2 and pMD2.G, were used for packaging plasmid. We co-transfected the 3 plasmids in 293FT cells for Lentivirus packaging and then collected the supernatant to harvest lentivirus to infect the MC38, CT26, and SW480 cells. After the infection, all 3 cell lines were selected with puromycin.

      DCLK1 Overexpression

      DCLK1 overexpression was achieved by transient transfection of the pLVX-IRES-DCLK1-Zsgreen plasmid into the HCT116 cell line using Neofect (Biotech Co, Ltd, Beijing, China) according to the manufacturer's instructions. The empty vector was used as a negative control.

      Subcutaneous Tumor Growth

      C57BL/6J, BALB/c, and nude mice were obtained from Charles River Laboratory (Beijing, China). In all experiments, 6- to 8-week-old mice were used. All mice were kept in a barrier facility at the animal center of Tsinghua University (Beijing, China). The animal facility in the laboratory has been licensed by the Association for Assessment and Accreditation of Laboratory Animal Care International. For the xenograft tumor model, 1 × 106 tumor cells were implanted SC into the right flanks of mice with 3 different genetic backgrounds. Tumor volume was measured every 4–5 days. The following formula was used to calculate the tumor volumes: V (mm3) = (length × width
      • Stein U.
      • Schlag P.M.
      Clinical, biological, and molecular aspects of metastasis in colorectal cancer.
      )/2. All animal experiments in this study were approved by the animal ethics committee.

      Establishment of Intestinal Carcinoma In Situ

      The tumor cells were adjusted to 3 × 107/mL and mixed with an equal volume of melted Matrigel (BD Biosciences) at a final concentration of 1.5 × 106 cells per 100 μL. Mice received 5% chloral hydrate intraperitoneally for anesthesia. The injection volume corresponded to mouse weight, that is, 200 μL chloral hydrate per 20-gram mouse. After anesthesia, we maintained the mice at a 37ºC constant-temperature operating table in an abdominal-upward position. An opening was made in the left lower abdomen of the mice to expose the colon, and 100 μL cell suspension coated with Matrigel (1.5 × 106 cells) was injected into the intestinal wall. After the injection, the abdominal incision was closed using a wound suture clamp. The anesthetized mice awakened 2 hours later, and their vital signs were observed daily. Three weeks later, the mice were killed and dissected to check the tumor growth.

      Tumor Growth After Rechallenge

      DCLK1-/- MC38 cells (1 × 106) were implanted SC into the left flank of C57BL/6J mice and DCLK1-/- CT26 cells were implanted in BALB/c mice. Tumor volumes were measured by caliper every 4–5 days. After the DCLK1-/- tumors regressed, DCLK1WT or DCLK1-/- tumor cells were implanted into the right abdomen of mice SC, and marked as the experimental group. We continued to observe the growth of tumor cells in the 2 groups. In the control group, the mice were implanted with only DCLK1WT or DCLK1-/- cells SC at 28 days, without any prior inoculation.

      CD4+, CD8+ T-Cell Depletion

      In vivo T-cell depletion was performed through intraperitoneal injection. Anti-CD4, anti-CD8 (clone GK1.5 and clone 53-6.7, respectively), and isotype control antibody were purchased from eBioscience. Before the SC injection of tumor cells, mice were injected with 150 μg antibody 4 times. After the mice were inoculated with tumor cells, the antibody was injected intraperitoneally at 3-day intervals to completely deplete the T cells. On day 8 after the initial antibody injection, each group, containing 6∼8 mice, was inoculated with 1 × 106 MC38 DCLK1WT and DCLK1-/- cells SC.

      Flow Cytometry Analysis of Immune Cell Populations

      For flow cytometry analysis, the tumor-bearing mice were killed by CO2 asphyxiation and the tumor tissues were removed on the seventh day after tumor inoculation. The tumor tissues were digested with Dulbecco’s modified Eagle medium/RPMI 1640 medium containing 2 mg/mL collagenase IV for 0.5 hours at 37ºC. Next, the tumor tissues were dissociated by gentleMACS Tissue Dissociator and run through a 70-μm cell strainer to obtain a single-cell suspension. Samples were centrifuged at 1000 rpm for 5 minutes twice and resuspended in fluorescence-activated cell sorter buffer. To stain intracellularly, we permeabilized the cell membrane with Foxp3/transcription factor staining buffer. All antibodies for flow cytometry population analysis are listed in Table 1, Table 2, Table 3. Nonviable cells were stained with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit and gated out. Experiments were performed using a BD Biosciences FACS Aria III, and results were analyzed using FlowJo software.
      Table 1T-Cell Subsets for Flow Cytometry Analysis to Define Immune Cell Populations
      PopulationMarkers
      CD4+ T cellsCD45+CD3+CD4+CD8-
      CD4+ effector T cellsCD45+CD3+CD4+CD8-CD44+CD62L-
      CD8+ T cellsCD45+CD3+CD4-CD8+
      CD8+ effector T cellsCD45+CD3+CD4-CD8+CD44+CD62L-
      MarkersFluorophore
      ViabilityAuqa
      CD45APC-eFluor780
      CD3APC
      CD4BV605
      CD8BV421
      CD44FITC
      CD62LPE
      APC, _______; FITC, fluorescein isothiocyanate; PE, ________.
      Table 2MDSC Subsets for Flow Cytometry Analysis to Define Immune Cell Populations
      PopulationMarkers
      MacrophageCD45+CD11b+F4/80+
      G-MDSCCD45+CD11b+F4/80-Gr1+Ly6G+
      M-MDSCCD45+CD11b+F4/80-Gr1+Ly6C+
      MarkersFluorophore
      ViabilityAuqa
      CD45APC-eFluor780
      CD11bAF700
      F4/80PE
      Gr1AF594
      Ly6GBV421
      Ly6CFITC
      APC, _______; FITC, fluorescein isothiocyanate; M-MDSC, monocytic myeloid-derived suppressor cell; PE, ________.
      Table 3NK and NKT Cells for Flow Cytometry Analysis to Define Immune Cell Populations
      PopulationMarkers
      NKCD45+CD3-NK1.1+
      NKTCD45+CD3+NK1.1+
      MarkersFluorophore
      ViabilityAqua
      CD45APC-eFluor780
      CD3APC
      NK1.1FITC
      APC, _______; FITC, fluorescein isothiocyanate; NKT, _______.

      CD8+ T-Cell Fluorescent Immunohistochemistry

      Seven days after the mice were inoculated, tumor-bearing mice were killed and tumor tissues were taken. All tissues were embedded in OCT cryostat sectioning medium and stored at -80°C. Before sectioning, tissue blocks were kept at -20ºC overnight. Frozen blocks were sectioned onto 5-μm–thick slides and kept in 1% paraformaldehyde at room temperature for approximately 3 minutes. After permeabilization with 0.5% Triton-X for 30 minutes, antigen retrieval was performed with glycine. Before staining, all slides were blocked for 1 hour at room temperature with 0.2% Tween-20 and 5% FBS to avoid nonspecific binding. Staining with primary antibodies was performed at 4ºC in moist dark chambers overnight. Primary antibodies were diluted in antibody diluent (1 × phosphate-buffered saline [PBS] + 0.2% Tween-20 + 5% FBS + 0.05% NaN3). The anti-mouse CD8α (clone 53-6.7) antibody was diluted at 1:100, rat IgG2a was used as a negative control (eBioscience) and diluted at 1:100. After staining with primary antibodies, we washed the slides with PBS for 10 minutes. Slides were incubated with secondary antibodies for more than 2 hours in a moist dark chamber at a dilution of 1:1000 (Alexa Fluor 488-AffiniPure goat anti-rabbit IgG; H+L). After removal of secondary antibodies, the slides were mounted with a mounting medium containing 4′,6-diamidino-2-phenylindole (100 ng/mL). After washing, slides were sealed with a coverslip and observed with laser scanning confocal microscopy (LSCM-FV1200). Three tumor sections were stained in each group.

      RNA Sequence Analysis

      Gene expression profiling

      RNA-seq libraries were sequenced on a BGISEQ-500. The yielded reads then were aligned to mm10 reference genome using STAR 2.6.0a,
      • Dobin A.
      • Davis C.A.
      • Schlesinger F.
      • Drenkow J.
      • Zaleski C.
      • Jha S.
      • Batut P.
      • Chaisson M.
      • Gingeras T.R.
      STAR: ultrafast universal RNA-seq aligner.
      with the following parameters-out Filter Multi map Nmax 20-out Filter Mismatch Nmax 999-out Filter Mismatch Nover Read Lmax 0.04–out Filter Intron Motifs Remove Noncanonical Unannotated–align Intron Min 20–align Intron Max 1000000-alignSJDBoverhangMin 1-sjdbOverhang 49 -sjdbScore 1 -quantMode Gene Counts. Aligned reads were quantified by STAR using GENCODE M20 as reference. The data and analysis pipeline were managed by BioQueue.
      • Yao L.
      • Wang H.
      • Song Y.
      • Sui G.
      BioQueue: a novel pipeline framework to accelerate bioinformatics analysis.
      To identify differentially expressed genes, we used edgeR,
      • Robinson M.D.
      • McCarthy D.J.
      • Smyth G.K.
      edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
      which also provided us depth-normalized read counts. Another R package, pheatmap, was used to visualize expression profiles.

      Enrichment Analysis

      We further performed both GO and KEGG enrichment analysis on these identified differentially expressed genes using cluster Profile.
      • Yu G.
      • Wang L.G.
      • Han Y.
      • He Q.Y.
      clusterProfiler: an R package for comparing biological themes among gene clusters.
      For GO enrichment, we considered as significant the ontology terms with q values smaller than 0.05 (adjusted by BH). For KEGG enrichment, we required significant pathways to have P values less than .05.

      Association Analysis

      Association analysis between DCLK1 and the MAPK-ERK pathway was performed with cor.test from R. Functional protein association networks between DCLK1 and proteins in the MAPK-ERK pathway was predicted by STRING (version 11),
      • Szklarczyk D.
      • Gable A.L.
      • Lyon D.
      • Junge A.
      • Wyder S.
      • Huerta-Cepas J.
      • Simonovic M.
      • Doncheva N.T.
      • Morris J.H.
      • Bork P.
      • Jensen L.J.
      • Mering C.V.
      STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.
      using an interaction score of 0.400 as a cut-off value. To validate our conclusion in human beings, we retrieved FPKM values of homogeneous genes from the TCGA-COAD project (n = 480) and calculated their correlation coefficients.

      Survival Analysis

      All of the messenger RNA expression data for TCGA data set analysis were downloaded from UCSC Xena (https://xenabrowser.net/datapages) in FPKM format. Samples with incomplete information or survival time shorter than 30 days were excluded and the optimal cut-off point of risk value was performed using the surv_cutpoint function in R package survminer to avoid bias.

      Mouse MDSCs (CD11b+Gr1+) Isolation and Co-culture With T Cells

      Subcutaneous DCLK1WT and DCLK1-/- tumors were dissected, digested with collagenase IV (Worthington, USA) at 37ºC, and filtered to a single-cell suspension. The EasySep Mouse MDSC (CD11b+ Gr1+) Isolation Kit was used to isolate MDSCs from tumor tissues. The purity of MDSCs, as indicated by flow cytometry, was greater than 70%. The corresponding spleens of DCLK1WT and DCLK1-/- groups were isolated, and the EasySep Mouse CD8+ T-Cell Isolation Kit was used to extract CD8+ T cells from the spleen of mice and stained with CFSE (10 μmol/L). The sorted MDSCs were co-cultured with CFSE-labeled T cells at a 1:1 ratio, and mouse CD3/CD28 (eBioscience) was added to activate the T cells. After 3 days, the proliferation of T cells was detected by flow cytometry.

      Multiplexed Immunofluorescence

      A colon cancer tissue microarray (HCol-Ade060CS1-01) containing 30 paired tissues of stage I/II intestinal cancer and matched paracancerous tissues was purchased from Shanghai Core Ultra. This tissue microarray was stained with the Opal 4 Multiplex reagents (PerkinElmer) containing DCLK1 (ab31704, 1:500) opal 690, CXCL1 (ab89318, 1:500) opal 570, and CD33 (ab269456, 1:500) opal 590, and then counterstained with 4′,6-diamidino-2-phenylindole. Multiplexed fluorophore-stained slides were photographed under an Olympus confocal microscope (LSCM-FV1200). The background was removed using plain white film and finally analyzed using the Form software.

      Western Blot Analysis

      DCLK1WT and DCLK1-/- tumor cells (1 × 106) were seeded in 60-mm dishes and cultured for 24 hours. Protease and phosphatase inhibitors (Beyotime) were dissolved in RIPA buffer. After the cells adhered, the culture supernatant was removed and the RIPA buffer was added for cell lysis. Cells were lysed with RIPA buffer on ice for half an hour. Total protein was extracted and concentrations were measured by BCA protein assay (Thermo Fisher Scientific). The Nuclear Extraction Kit was used for cytoplasmic and nuclear protein isolation. The protein samples were applied to 8%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). After 8% nonfat milk blockade for 1 hour, the membrane was incubated with primary antibodies at 4ºC overnight. Primary antibodies included anti-DCLK1 (1:1000, cat# 62257; Cell Signaling Technology, and cat# 31704; Abcam), anti-ERK1/2 (1:1000, cat# 4695; Cell Signaling Technology), anti–phospho-ERK (1:1000, cat# 9101; Cell Signaling Technology), c-Myc (1:1000, CST#18583; Cell Signaling Technology), lamin A/C (1:1000, cat# 4777; Cell Signaling Technology), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, cat# 5174; Cell Signaling Technology). After the polyvinylidene difluoride membrane was washed 3 times by 1× Tris-buffered saline with Tween-20, the secondary antibodies were incubated for 1 hour at room temperature. The membrane was developed with the Bio-Rad imaging system.

      RNA Extraction and Quantitative Real-Time PCR

      Total RNA was extracted using TRIzol reagent (Invitrogen). To prepare complementary DNA using the PrimeScript RT reagent Kit (RR047A; TaKaRa), 1 μg total RNA was reverse-transcribed. Expression of specific genes was verified using the SYBR Green system (Applied Biosystems), normalized with GAPDH. In the RT-PCR system, all PCR reactions contained forward and reverse primers (100 pmol). The RT-PCR reaction was performed on a 7500 Sequence Detection System (Applied Biosystems). With the comparative Ct method, we measured the relative gene expression changes (X = 2-ΔΔCT). Sequences of quantitative PCR primers are listed as follows: CXCL1 of mice: 5’-CTGGGATTCACCTCAAGAACATC-3’ and 5’-CAGGGTCAAGGCAAGCCTC-3’; CXCL1 of human beings: 5’-TCCTGCATCCCCCATAGTTA-3’ and 5’-CTTCAGGAACAGCCACCAGT-3’; CXCL2 of mice: 5’-CCAACCACCAGGCTACAGG-3’ and 5’-GCGTCACACTCAAGCTCTG-3’; CXCL2 of human beings: 5’-CCCATGGTTAAGAAAATCATCG-3’ and 5’-CTTCAGGAACAGCCACCAAT-3’; GAPDH of mice: 5’-ATCAAGAAGGTGGTGAAGCA-3’ and 5’-AGACAACCTGGTCCTCAGTGT-3’; GAPDH of human beings: 5’-ACAACTTTGGTATCGTGGAAGG-3’ and 5’-GCCATCACGCCACAGTTTC-3’.

      ELISA Assays

      The tumor cells were seeded evenly on a 6-well plate for 24 hours. After that, cell culture supernatant was collected. To remove cell fragments, the supernatant was centrifuged at 16,000 × g for 10 minutes. The mouse CXCL1 and CXCL2 expression levels were measured by mouse Quantikine ELISA kits (MKC00B/MM200; R&D Systems), and the human CXCL1 and CXCL2 expression levels were measured by the Human SimpleStep ELISA Kit (ab190805/ab184862; Abcam). All of the experimental methods were performed according to the manufacturer’s protocols.

      Cell Proliferation and Migration Assays

      The cell proliferation experiments were performed with the Cell Counting Kit-8 kit (Dojindo, Japan). The DCLK1WT and DCLK1-/- cells were seeded on a 96-well plate, and the absorbance was measured at 450-nm wavelength by a microplate reader for 4–5 days. Then, the proliferation curve was calculated.
      Cells were seeded in the upper chamber of a 24-well, 8.0-μm pore membrane chamber (Corning, Inc, Corning, NY) with FBS-free medium (1 × 105 cells for MC38 and 5 × 104 for CT26), followed by the addition of 600 μL medium containing 10% FBS into the lower chamber. The MC38 and CT26 cells were cultured for 24 hours for migration. After 24 hours, we removed the nonmigrating cells from the upper chamber and fixed the chamber membranes with 4% PFA for 10 minutes. The migrating cells were stained with crystal violet (0.1%) for 5 minutes, and stained cells were counted under the microscope.

      Colony Formation Assays

      MC38 and CT26 cells (2 × 102/well) were seeded in 6-well plates, and the clone formation was detected under the microscope every 2∼3 days. Two weeks later, we removed the culture media and washed with PBS twice, and then fixed with 4% PFA for 10 minutes, stained with crystal violet. Each clone contained more than 50 cells.

      Statistical Analysis

      All of the data are presented as means ± SD. Two groups were compared using the Student t test. Data were analyzed with GraphPad Prism (version 9.0), and a P value < .05 was considered significant. Each independent experiment was repeated more than 3 times.

      CRediT Authorship Contributions

      Rui yan, MD (Conceptualization: Equal; Data curation: Equal)
      Jianjian Li (Data curation: Equal)
      Zeru Xiao (Data curation: Supporting; Methodology: Supporting)
      Xiaona Fan (Methodology: Supporting)
      Heshu Liu (Formal analysis: Supporting; Methodology: Supporting)
      Ying Xu (Formal analysis: Supporting)
      Ruya Sun (Writing – original draft: Supporting)
      Jian Liu (Writing – review & editing: Supporting)
      Jiannan Yao (Formal analysis: Supporting)
      Guangyu An (Supervision: Supporting)
      Yan Shi (Writing – review & editing: Supporting)
      Yang Ge (Conceptualization: Equal; Supervision: Equal)

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