Abstract

   To explore the specific molecular mechanisms of Siglec6 CAR-T therapy
   in acute myeloid leukemia (AML). AML samples were selected from the GEO
   database for bioinformatics analysis. Siglec6 was knocked down and
   overexpressed in MOLM-13 cells through lentiviral infection, and then
   injected into NOD/SCID mice via the tail vein to detect the
   distribution of AML in mice using in vivo imaging. MOLM-13 cells were
   divided into six groups: oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1,
   oe-Siglec6 + Dasatinib, oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 neutralizing antibody. The
   expression levels of related proteins were detected by Western blot and
   immunofluorescence, and the cell invasion and proliferation abilities
   were tested by Transwell and CCK8 assays. Finally, MOLM-13 cells were
   co-cultured with CD19-CAR-T and Siglec6-CAR-T cells, and the apoptosis
   level of MOLM-13 cells after co-culture was detected by flow cytometry.
   In vivo imaging found that after overexpression of Siglec6 in MOLM-13
   cells, AML cells were more widely distributed in mice; Western blot and
   immunofluorescence detected the protein levels in AML cells and found
   that compared with the oe-NC group, the expression levels of Siglec6,
   p-SHP2, IL-3, and p-ERK1/2 proteins were increased in the oe-Siglec6
   group; cell invasion, migration, and proliferation abilities were
   enhanced, and these abilities were reversed after treatment with SHP2
   inhibitors, Src inhibitors, SHP2 agonists, and IL-3 neutralizing
   antibodies. Finally, both in vitro and in vivo, it was found that
   compared with CD19 CAR-T, the apoptosis level of AML cells treated with
   Siglec6 CAR-T was increased, and their distribution in mice was
   reduced. Siglec6 CAR-T reduces the proliferation, invasion, and
   migration abilities of AML cells by acting on the SHP2/Src/ERK/IL-3
   axis.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-00456-x.

   Keywords: CAR-T, AML, Siglec6, SHP2, Src, IL-3, ERK

   Subject terms: Cell biology, Molecular medicine

Introduction

   Acute myeloid leukemia (AML) is the most common type of acute leukemia
   in adults. It is characterized by the malignant transformation and
   uncontrolled proliferation of myeloid hematopoietic progenitor cells,
   leading to the accumulation of a large number of immature cells in the
   bone marrow and peripheral blood. Standard induction chemotherapy can
   achieve a complete remission rate of 60–80% in AML. However,
   ultimately, 50–70% of AML patients still relapse, and the 5-year
   survival rate is only 20–30%^[42]1,[43]2. Chemotherapy resistance and
   short-term relapse remain the main factors threatening patient
   survival^[44]3. Although the survival of acute leukemia has been
   extended with the application of hematopoietic stem cell
   transplantation and small molecule inhibitors, the 5-year survival rate
   of AML patients after treatment is still low due to the high relapse
   rate of AML. Therefore, there is an urgent need for a new treatment
   method for AML^[45]4.

   In recent years, CAR T-cell (CAR-T) technology has attracted widespread
   attention in the treatment of AML. Some specific antigens expressed on
   the surface of AML cells, such as CD19, CD33, CD123, etc., provide the
   possibility for precise recognition and targeted treatment of CAR-T
   cells^[46]5. Through the design of chimeric antigen receptors, CAR-T
   cells can effectively activate T cells, recognize and kill AML cells,
   thereby theoretically achieving targeted treatment of the
   disease^[47]6. However, although CAR-T cell therapy has achieved
   significant efficacy in some hematological malignancies, its
   application in AML has not been ideal, often facing low cure rates and
   high relapse risks^[48]7. This is mainly related to the heterogeneity
   of AML cells and their adaptive changes to treatment. At the same time,
   the expression of antigens on the surface of AML cells also has certain
   variability, which limits the recognition and killing of target cells
   by CAR-T cells. Therefore, to improve the effectiveness of CAR-T cells
   in the treatment of AML, it is urgent to explore the possibility of
   targeting other AML-specific antigens.

   Siglec-6 (sialic acid-binding immunoglobulin-like lectin-6) is an
   immunomodulatory cell surface receptor belonging to the Siglec
   family^[49]8. It consists of three extracellular immunoglobulin (Ig)
   domains and two intracellular immunoreceptor tyrosine-based inhibitory
   motifs (ITIMs), and its molecular structure is very similar to that of
   Siglec-3 (CD33)^[50]9. Siglec-6 is expressed in AML, but not detected
   in normal hematopoietic stem cells and progenitor cells^[51]10. This
   allows CAR-T cells to accurately recognize and kill AML cells by
   targeting Siglec6 on AML, demonstrating enormous clinical
   potential^[52]11. Therefore, we have conducted in-depth research on the
   Siglec-6 target in AML in order to seek more effective treatment
   strategies.

   SHP2 is an important tyrosine phosphatase encoded by the PTPN11 gene
   and is a key regulatory factor in cellular signal transduction
   pathways^[53]12. SHP2 plays a crucial role in various cellular
   processes, including cell proliferation, differentiation, and survival.
   This protein contains two SH2 domains that can specifically bind to
   phosphorylated tyrosine residues, thereby transmitting signals within
   cells. The main function of SHP2 is dephosphorylation, which can
   regulate various intracellular signaling pathways, such as cell growth
   factor receptors, integrins, and immune receptor signaling pathways. By
   removing tyrosine phosphate groups, SHP2 plays a negative regulatory
   role, promoting the termination or regulation of downstream signals. It
   has also been shown that there is a direct interaction between Siglec6
   and SHP2. Siglec6 inhibits downstream signaling pathways, such as Src,
   IL-3, and ERK signaling pathways, by recruiting SHP2. This interaction
   is critical for understanding the mechanism of Siglec6 CAR T cells.
   Previous studies may have reported aberrant expression or function of
   SHP2 in AML. Therefore, we speculate that SHP2 plays a key role in AML,
   and its abnormal expression or activation is closely related to the
   development of AML. In-depth exploration of the molecular mechanisms
   affecting SHP2 expression levels in AML may provide new strategies for
   the treatment of AML.

Materials and methods

Preparation of CAR-T cells

   First, we prepared the CAR component of CAR-T cells. Siglec6-CAR
   consists of a CD8α signal peptide, a human anti-Siglec6 scFv, a CD8α
   hinge, a CD8α transmembrane, a 4-1BB intracellular co-stimulatory
   domain, and an intracellular CD3ζ domain. The control CD19-CAR is
   composed of a CD8α signal peptide, a human anti-CD19 scFv, a CD8α
   hinge, a CD8α transmembrane, a 4-1BB intracellular co-stimulatory
   domain, and an intracellular CD3ζ domain. Polyethyleneimine (PEI) was
   used to co-transfect HEK-293T with CAR vectors and two packaging
   plasmids (pMD.2G and psPAX2). After transfection, the supernatant was
   collected at 48 and 72 h, filtered through a 0.45 μm filter
   (Millipore), and then centrifuged at 100,000×g for 120 min at 4 °C. The
   concentrated virus was resuspended and stored at − 80 °C. Lentivirus
   titration was performed using the TCID50 method.

   For T cell preparation, we purchased peripheral blood mononuclear cells
   (PBMCs) from Hunan Fenghui Bio, purified them by Ficoll-Paque PLUS (GE
   Healthcare) density gradient, and stored them frozen. On the day before
   CAR-T cell production, PBMCs were thawed at 37 °C and incubated
   overnight in RPMI 1640 with 10% FBS, penicillin, streptomycin,
   amphotericin B, and L-glutamine at 37 °C and 5% CO2. The next day,
   PBMCs were activated with a 1:1 ratio of CD3:CD28 beads (Invitrogen)
   for 40 to 48 h. Subsequently, the CAR construct was transferred into
   activated T cells through lentiviral infection to produce CD19-CAR-T
   and Siglec6-CAR-T.

AML cell culture, infection, and treatment

   MOLM-13 cells, purchased from Procell, were cultured in RPMI1640 medium
   (11875093, Thermo Fisher) containing 10% fetal bovine serum (A5256701,
   Thermo Fisher). The cells were authenticated by Short Tandem Repeat
   (STR) and confirmed to be free of contamination. They were routinely
   cultured in a 37 °C, 5% CO2 incubator. Cells were passaged every 2–3
   days, and cells in the logarithmic growth phase were used for
   subsequent experiments. To infect MOLM-13 cells with lentivirus, they
   were seeded into 24-well plates the day before infection and grown to
   50% confluence. The cells were then transduced with lentiviral vectors
   at a multiplicity of infection (MOI) of 10 using lentivirus oe-Siglec6
   (2 × 10^9 TU/ml) and sh-Siglec6 (2 × 10^9 TU/ml) from Yunzhou Bio, with
   a control vector for comparison. After 48 h, cells were selected with
   puromycin (2 µg/mL) for two weeks.

   A portion of MOLM-13 cells were treated with 10 µg/mL of SHP2 inhibitor
   (PHPS1, MCE), 10 µg/mL of Src inhibitor (S1021, Selleck), and 5 µg/mL
   SHP2 agonist (75330-75-5, Merck) for 12 h to promote and inhibit
   protein expression levels. Another portion of MOLM-13 cells were
   co-cultured with CAR-T cells at a 1:3 ratio to investigate the killing
   effect of CAR-modified T cells on AML cells.

Animal modeling and in vivo imaging

   Two-week-old, 16–18 g NOD/SCID mice, purchased from Vital River, were
   bred under specific pathogen-free conditions. Nude mice were randomly
   divided into five groups: oe-NC, oe-NC + CAR-T, oe-Siglec6,
   oe-Siglec6 + CAR-T, and oe-Siglec6 + Siglec6 CAR-T, with six mice per
   group. Luc-labeled MOLM-13 AML cells in the logarithmic growth phase
   were centrifuged, counted, and adjusted to a cell density of
   1 × 10^7/mL with PBS. MOLM-13 cells were injected into mice via the
   tail vein. Tumor implantation was confirmed 25 days later.
   Subsequently, treated CD19-CAR-T and Siglec6-CAR-T cells were injected
   into the mice, and disease progression was monitored and tumor volume
   measured using bioluminescence imaging on day 28. Mice were
   anesthetized with 2% isoflurane in a nose cone and maintained under
   inhalation anesthesia. D-luciferin (Goldbio, 150 mg/kg) was
   administered intraperitoneally 10 min before scanning. After the
   experiment, mice were euthanized by CO2 inhalation. All experimental
   protocols were approved by the Ethics Committee of Tianjin Jinke Bona
   Biotechnology Co. A statement that all methods have been performed in
   accordance with relevant guidelines and regulations and are reported in
   accordance with ARRIVE guidelines.

Western blot

   Collected MOLM-13 cells that had been treated with lentiviral infection
   and protein agonists and antagonists, as well as IL-3 monoclonal
   antibody, were lysed with RIPA lysis buffer (P0013B, Beyotime)
   containing the protease inhibitor PMSF (ST506, Beyotime) to extract
   total protein. Protein quantification was performed using the BCA assay
   kit. Proteins were separated by SDS-PAGE electrophoresis, transferred
   to PVDF membranes. At room temperature and closed with 5% skimmed milk
   for 2 h. The membranes were then incubated with primary antibodies
   against Siglec6 (1:1000, abcam), p-SHP2 (1:500, abclonal), IL-3
   (1:1000, abcam), p-ERK1/2 (1:500, Thermofisher), MMP2 (1:1000, abcam),
   MMP9 (1:500, abclonal), CyclinA1 (1:2000, Thermofisher), CyclinD1
   (1:1000, abclonal), Ki-67 (1:1000, abcam), cleaved-Caspase-3 (1:1000,
   abcam), cleaved-Caspase-9 (1:1000, abcam) and GAPDH (1:2000, abcam).
   HRP-conjugated goat anti-rabbit and goat anti-mouse secondary
   antibodies (1:5000, abcam) were added and incubated for 1 h at room
   temperature. The membranes were washed with TBST, and ECL
   chemiluminescence reagent was used for development. Quantitative
   analysis was performed using Image J software.

Immunofluorescence

   Cell culture medium was removed, and cells were fixed with 4%
   paraformaldehyde at room temperature for 20 min. After washing with
   phosphate buffer saline (PBS), cells were permeabilized with PBS
   containing 0.1% Triton X100 for 5 min, followed by blocking with 10%
   skim milk for 1 h to block non-specific antibody binding sites. Cells
   were then incubated overnight at 4 °C with Siglec6 (1:1000, abcam) and
   p-SHP2 (1:100, abcam) antibodies. After washing three times with PBS,
   goat anti-rabbit and donkey anti-mouse secondary antibodies were added
   and incubated for 1 h at room temperature. Finally, DAPI was used to
   counterstain the cell nuclei. DAPI was diluted to a concentration of
   1 µg/mL with PBS. The diluted DAPI was added and incubated for 10 min
   at room temperature. Cells were washed 3 times with PBS for 5 min each
   to remove unbound DAPI. Images were visualized using an inverted
   fluorescence microscope (OLYMPUS, IX73) and quantified using imageJ.

CCK8 analysis

   MOLM-13 cells were collected after 12, 24, and 48 h of treatment. The
   culture medium was removed from the wells, and 10µL of CCK-8 solution
   was added to each well. The plates were incubated for 2 h at 37 °C and
   5% CO2. Absorbance (A) values at 450 nm were measured using a
   microplate reader.

Transwell invasion assay

   MOLM-13 cells were seeded at a density of 5 × 10^4/mL in the upper
   chamber of a Transwell plate pre-coated with Matrigel. 600 µL of
   culture medium containing 20% fetal bovine serum was added to the lower
   chamber. After 24 h, the lower chamber was washed twice with PBS, and
   cells were fixed with 4% paraformaldehyde for 30 min and stained with
   0.1% crystal violet solution for 20 min. Quantitative analysis was
   performed using Image J software in six random fields of view.

Flow cytometry for apoptosis

   CAR-T and MOLM-13 cells were separated using Ficoll-Paque. The MOLM-13
   cell suspension was centrifuged for 5 min and washed three times with
   PBS. Cells were stained with 500 µl of binding buffer and gently mixed,
   followed by the addition of 5 µl of PI and 5 µl of Annexin V (C1062S,
   Beyotime) and thorough mixing. Cells were incubated at room temperature
   for 15 min. Apoptosis levels were detected by flow cytometry after 1 h
   and quantified using FlowJo software (version FlowJo v10.9,
   [54]https://www.flowjo.com/). The experiments were repeated three
   times.

Bioinformatics analysis of AML

   Downloaded datasets related to acute myeloid leukemia, [55]GSE10358 and
   [56]GSE24395, from the Gene Expression Omnibus database
   ([57]https://www.ncbi.nlm.nih.gov/geo/). First, background correction
   and normalization were performed using the Robust Multi-array Average
   (RMA) (version 1.70.0,
   [58]https://bioconductor.org/packages/release/bioc/html/affy.html)
   method, which is a standard step in processing microarray data. RMA not
   only effectively removes background noise but also enhances the
   comparability of data across different chips. Next, the Combat (version
   3.40.0,
   [59]https://bioconductor.org/packages/release/bioc/html/sva.html)
   method was applied for batch effect correction, which can eliminate
   technical variations between different experimental batches, ensuring
   the accuracy of subsequent analyses. In the differential expression
   analysis, the limma package (version 3.46.0,
   [60]https://bioconductor.org/packages/release/bioc/html/limma.html) was
   utilized to efficiently identify genes with significant expression
   differences under different conditions. The selection criteria were set
   to |log2 Fold Change|>1 and p-value < 0.05, ensuring that the selected
   genes are biologically significant. Further multiple hypothesis testing
   was corrected using the Benjamini-Hochberg method to control the false
   positive rate. By using the ggplot2 package (version 3.3.5,
   [61]https://ggplot2.tidyverse.org) for visualizing the results of the
   differential analysis, the expression patterns of significantly
   different genes can be intuitively displayed. This visualization not
   only aids in the interpretation of results but also provides a
   foundation for subsequent biological analyses. Among the identified
   differentially expressed genes, the ClusterProfiler package (version
   3.18.1,
   [62]https://bioconductor.org/packages/release/bioc/html/clusterProfiler
   .html) was used for GO and KEGG pathway enrichment analysis, revealing
   the roles of these genes in cellular components, molecular functions,
   and biological processes. Additionally, KEGG pathway analysis helps to
   understand the role of these genes in key biological pathways, thereby
   providing clues for potential treatment strategies for AML. Finally, by
   selecting the Spearman statistical method, a correlation analysis (R
   version 4.1.0, [63]https://www.r-project.org) of differential genes was
   performed to discover potential associations between gene variables.

Statistical analysis

   Statistical analysis was performed using GraphPad Prism 8.0.2.
   Statistical significance was assessed using analysis of variance
   (ANOVA) or t-tests, with Tukey’s post-hoc test. A P-value of less than
   0.05 was considered statistically significant. All results are
   expressed as the mean ± standard error of the mean (SEM). Cellular
   experiments were independently repeated three times, and animal
   experiments were conducted with six animals per group.

Results

   1. Bioinformatics Analysis of AML.

   Through analysis with the limma package, a total of 397 significantly
   differentially expressed genes (DEGs) were identified in the
   [64]GSE10358 dataset, with 238 upregulated and 159 downregulated genes;
   the [65]GSE24395 dataset had a total of 647 DEGs, with 415 upregulated
   and 232 downregulated genes. The upregulation and downregulation of
   these genes indicate significant transcriptional changes in AML cells
   (Fig. [66]1A). Intersection analysis revealed 52 common genes
   (Fig. [67]1B). These common genes were further analyzed using GO and
   KEGG analyses, which showed significant enrichment results suggesting
   that these DEGs play important roles in cellular components, molecular
   functions, and biological processes. For instance, DEGs were enriched
   in cellular components related to the cell membrane, potentially
   associated with cell signaling and cell-cell interactions.
   Additionally, the enrichment results of molecular functions indicated
   that these genes may be involved in important biological functions such
   as enzymatic activity and binding activity, highlighting their key
   roles in cellular metabolism and proliferation (Fig. [68]1C). KEGG
   pathway analysis results indicated that DEGs were significantly
   enriched in the MAPK signaling pathway, PI3K-Akt signaling pathway, and
   Ras signaling pathway, which are closely related to tumor cell growth,
   apoptosis, and migration (Fig. [69]1D). Particularly, the MAPK
   signaling pathway is a key regulatory pathway in many types of cancer,
   and its activation is often associated with tumor invasiveness and drug
   resistance. Spearman correlation analysis revealed a positive
   correlation between Src and SHP2 (PTPN11), suggesting a potential
   synergistic role of these two in signal transduction (Fig. [70]1E). The
   positive correlation between SHP2 (PTPN11) and ERK1/2 (MAPK1) further
   supports the importance of the MAPK pathway in AML (Fig. [71]1F). The
   positive correlation between IL-3 and Siglec6 may reveal a potential
   role of IL-3 in AML cell proliferation, and Siglec6, as an
   immunomodulatory molecule, its expression may affect the
   microenvironment of AML (Fig. [72]1G).

Fig. 1.

   [73]Fig. 1
   [74]Open in a new tab

   Bioinformatics Analysis of AML. (A) Volcano plots of differential genes
   in the [75]GSE10358 and [76]GSE24395 datasets; (B) Venn diagram of
   differential genes; (C) Bar chart of GO enrichment analysis; (D) Bubble
   chart of KEGG enrichment analysis; (E) Scatter plot of the correlation
   between Src and SHP2 (PTPN11); (F) Scatter plot of the correlation
   between SHP2 (PTPN11) and ERK1/2 (MAPK1); (G) Scatter plot of the
   correlation between IL-3 and Siglec6.

   2. In Vivo Experiments Demonstrate that High Expression of Siglec-6
   Promotes the Distribution and Content Level of AML.

   MOLM-13 cells infected with lentivirus were injected into NOD/SCID mice
   via the tail vein, divided into oe-NC, oe-Siglec6, sh-NC, sh-Siglec6
   four groups, and the distribution of AML in mice was detected by in
   vivo imaging. It was found that compared with the oe-NC group, the AML
   content in the oe-Siglec6 group was higher and more widely distributed;
   compared with the sh-NC group, the AML content and distribution in the
   sh-Siglec6 group were both weakened (Fig. [77]2A, B).

Fig. 2.

   [78]Fig. 2
   [79]Open in a new tab

   In Vivo Experiments Demonstrate that High Expression of Siglec-6
   Promotes the Distribution and Content Level of AML. (A) In vivo imaging
   technology detects the tumor formation of AML after treatment with
   oe-NC, oe-Siglec6, sh-NC, sh-Siglec6 in mice; (B) Statistical analysis
   of in vivo imaging technology detecting the tumor formation of AML
   after treatment with oe-NC, oe-Siglec6, sh-NC, sh-Siglec6 in mice. Data
   are expressed as mean ± standard deviation. N = 6; **P < 0.01.

   3. Siglec6 Acts on AML through the SHP2/Src/ERK/IL-3 Axis.

   MOLM-13 cells were divided into six groups: oe-NC, oe-Siglec6,
   oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody. Western
   blot was used to detect the protein expression levels of Siglec6,
   p-SHP2, IL-3, pERK1/2, and immunofluorescence was used to detect the
   fluorescence levels of Siglec6 and p-SHP2. It was found that compared
   with the oe-NC group, the protein expression levels of Siglec6, p-SHP2,
   IL-3, pERK1/2, and the fluorescence levels of Siglec6 and p-SHP2 in the
   oe-Siglec6 group were increased; compared with the oe-Siglec6 group,
   the protein expression levels of Siglec6, p-SHP2, IL-3, pERK1/2, and
   the fluorescence levels of Siglec6 and p-SHP2 in the oe-Siglec6 + PHPS1
   group were weakened, and the same was true for the
   oe-Siglec6 + Dasatinib group; compared with the oe-Siglec6 + Dasatinib
   group, the protein expression levels of Siglec6, p-SHP2, IL-3, pERK1/2,
   and the fluorescence levels of Siglec6 and p-SHP2 in the
   oe-Siglec6 + Dasatinib + Lovastatin group were enhanced; while compared
   with the oe-Siglec6 + Dasatinib + Lovastatin group, the protein
   expression level of p-SHP2 and the fluorescence level of p-SHP2 in the
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody group
   remained unchanged, the protein expression levels of Siglec6, IL-3,
   pERK1/2 were weakened, and the fluorescence level of Siglec6 was
   weakened (Fig. [80]3A, B). This proves that Siglec6 acts on AML through
   the SHP2/Src/ERK/IL-3 axis.

Fig. 3.

   [81]Fig. 3
   [82]Open in a new tab

   Siglec6 Acts on AML through the SHP2/Src/ERK/IL-3 Axis. (A) Western
   blot detects the protein expression levels of Siglec6, p-SHP2, IL-3,
   pERK1/2 in AML cells in the oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1,
   oe-Siglec6 + Dasatinib, oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups;
   GAPDH as control protein; (B) Immunofluorescence detects the protein
   expression levels of Siglec6 and p-SHP2 in the oe-NC, oe-Siglec6,
   oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups.
   Data are expressed as mean ± standard deviation. N = 3; *P < 0.05;
   **P < 0.01; ^nsP>0.05.

   4. Siglec6 Promotes the Proliferation and Invasion-Metastasis Abilities
   of AML through the SHP2/Src/ERK/IL-3 Axis.

   MOLM-13 cells were divided into six groups: oe-NC, oe-Siglec6,
   oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody. Western
   blot was used to detect the expression levels of invasion-related
   proteins MMP2 and MMP9, and proliferation-related proteins CyclinA1,
   CyclinD1 and Ki-67. Transwell assays were used to test cell migration
   ability, and CCK8 assays were used to test cell activity. It was found
   that compared with the oe-NC group, the oe-Siglec6 group showed
   increased expression levels of MMP2, MMP9, CyclinA1, CyclinD1 and Ki-67
   proteins, cell migration ability, and cell activity. Compared with the
   oe-Siglec6 group, the oe-Siglec6 + PHPS1 group showed decreased
   expression levels of MMP2, MMP9, CyclinA1, CyclinD1 and Ki-67 proteins,
   cell migration ability, and cell activity. Similarly, the
   oe-Siglec6 + Dasatinib group also showed decreased levels of these
   proteins, cell migration ability, and cell activity. Compared with the
   oe-Siglec6 + Dasatinib group, the oe-Siglec6 + Dasatinib + Lovastatin
   group showed increased expression levels of MMP2, MMP9, CyclinA1,
   CyclinD1 and Ki-67 proteins, cell migration ability, and cell activity.
   In contrast, compared with the oe-Siglec6 + Dasatinib + Lovastatin
   group, the oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal
   antibody group showed decreased expression levels of MMP2, MMP9,
   CyclinA1, CyclinD1 and Ki-67 proteins, cell migration ability, and cell
   activity (Fig. [83]4A-D).This demonstrates that Siglec6 promotes the
   proliferation and invasion-metastasis abilities of AML through the
   SHP2/Src/ERK/IL-3 axis.

Fig. 4.

   [84]Fig. 4
   [85]Open in a new tab

   Siglec6 Promotes the Proliferation and Invasion-Metastasis Abilities of
   AML through the SHP2/Src/ERK/IL-3 Axis. (A) Western blot detects the
   expression levels of MMP2, MMP9, CyclinA1, CyclinD1 proteins in AML
   cells in the oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1,
   oe-Siglec6 + Dasatinib, oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups;
   (B) Transwell assay detects the invasion ability of AML cells in the
   oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups;
   (C) CCK8 assay detects the proliferation ability of AML cells in the
   oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups;
   (D) Western blot detects the expression levels of Ki-67 in AML cells in
   the oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 monoclonal antibody groups;
   GAPDH as control protein; Data are expressed as mean ± standard
   deviation. N = 3; *P < 0.05; **P < 0.01; ^nsP>0.05.

   5. In Vitro and In Vivo Experiments Demonstrate that Siglec6 CAR-T
   Inhibits the Progression of AML.

   MOLM-13 cells were co-cultured with CAR-T cells and divided into five
   groups: oe-NC, oe-NC + CAR-T, oe-Siglec6, oe-Siglec6 + CAR-T, and
   oe-Siglec6 + Siglec6 CAR-T. Firstly, the results of Western blot
   experiments showed that the expression levels of cleaved-Caspase-3 and
   cleaved-Caspase-9 were significantly higher in the oe-NC + CAR-T group
   compared to the oe-NC group, while the expression levels of
   cleaved-Caspase-3 and cleaved-Caspase-9 in the oe-Siglec6 group showed
   no significant changes. Caspase-9 expression levels did not change
   significantly. The expression levels of cleaved-Caspase-3 and
   cleaved-Caspase-9 were significantly elevated in the oe-Siglec6 + CAR-T
   group compared to the oe-Siglec6 group. The expression levels of
   cleaved-Caspase-3 and cleaved-Caspase-9 were further increased in the
   oe-Siglec6 + Siglec6 CAR-T group compared to the oe-Siglec6 + CAR-T
   group (Fig. [86]5A). Apoptosis levels of AML cells were detected by
   flow cytometry. It was found that compared with the oe-NC group, the
   oe-NC + CAR-T group showed increased apoptosis levels of AML cells,
   while the oe-Siglec6 group showed no significant change in apoptosis
   levels. Compared with the oe-Siglec6 group, the oe-Siglec6 + CAR-T
   group showed increased apoptosis levels of AML cells. Compared with the
   oe-Siglec6 + CAR-T group, the oe-Siglec6 + Siglec6 CAR-T group showed
   further increased apoptosis levels of AML cells (Fig. [87]5B). In vivo
   experiments were conducted using NOD/SCID mice. MOLM-13 cells coupled
   with luciferase were injected into mice, and CAR-T treatment was
   administered, also divided into five groups: oe-NC, oe-NC + CAR-T,
   oe-Siglec6, oe-Siglec6 + CAR-T, and oe-Siglec6 + Siglec6 CAR-T. In vivo
   imaging detected the distribution of AML and found that compared with
   the oe-NC group, the oe-NC + CAR-T group showed weakened distribution
   of AML. The oe-Siglec6 group showed no significant change in AML
   distribution. Compared with the oe-Siglec6 group, the
   oe-Siglec6 + CAR-T group showed weakened distribution of AML cells.
   Compared with the oe-Siglec6 + CAR-T group, the oe-Siglec6 + Siglec6
   CAR-T group showed further weakened distribution of AML cells
   (Fig. [88]5C). The above results suggested that Siglec6 CAR-T could
   target Siglec6 on AML and act on SHP2/Src/IL-3/ERK axis to inhibit
   tumor progression (Fig. [89]6).

Fig. 5.

   [90]Fig. 5
   [91]Open in a new tab

   In Vitro and In Vivo Experiments Demonstrate that Siglec6 CAR-T
   Inhibits the Progression of AML. (A) Western blot detects the
   expression levels of cleaved-Capase-3 and cleaved-Capase-9 in AML cells
   in the oe-NC, oe-NC + CAR-T, oe-Siglec6, oe-Siglec6 + CAR-T, and
   oe-Siglec6 + Siglec6 CAR-T groups after co-culturing with CAR-T cells;
   GAPDH as control protein; (B) Flow cytometry detects the apoptosis
   levels of MOLM-13 cells in the oe-NC, oe-NC + CAR-T, oe-Siglec6,
   oe-Siglec6 + CAR-T, and oe-Siglec6 + Siglec6 CAR-T groups after
   co-culturing with CAR-T cells; C: In vivo experiments detect the
   distribution of MOLM-13 cells in the oe-NC, oe-NC + CAR-T, oe-Siglec6,
   oe-Siglec6 + CAR-T, and oe-Siglec6 + Siglec6 CAR-T groups. Data are
   expressed as mean ± standard deviation. N = 3; *P < 0.05; **P < 0.01;
   ^nsP>0.05.

Fig. 6.

   [92]Fig. 6
   [93]Open in a new tab

   Siglec6 CAR T cells suppressed progression of AML via inhibiting
   Siglec6/ SHP2/ Src/ IL-3/ ERK signaling.

Discussion

   AML is a heterogeneous disease with diverse genomic alterations and
   mutations. Significant therapeutic advancements have been made through
   large-scale genomic assessments, providing detailed insights into AML’s
   molecular structure^[94]13. However, primary resistance and disease
   relapse remain major challenges, affecting more than half of AML
   patients due to diverse clones and somatic heterogeneity. CAR-T
   therapy, targeting antigens like CD33, CD123, CLL-1, and NKG2DLs, shows
   promise but faces issues such as poor persistence, insufficient
   efficacy, and target escape^[95]14. Therefore, finding new CAR-T
   targets is a top priority. Siglec-6 is an immunoglobulin-like lectin
   that belongs to the CD33-related subgroup of Siglecs and is mainly
   expressed in the placenta, mast cells, and memory B cells^[96]15. It is
   also highly expressed in AML. Interestingly, Siglec-6 is normally
   expressed in normal myeloid cells, suggesting that Siglec-6 may play an
   important role in the diagnosis and treatment of AML and has the
   potential to be a potential target for CAR-T. Therefore, we have
   explored the feasibility of Siglec6 as an AML target and its molecular
   mechanisms in depth.

   We first intervened with Siglec6 in AML through lentiviral infection
   and selected NOD/SCID mice to explore the relationship between the
   expression level of Siglec6 in AML and the progression of AML through
   in vivo imaging. After treating the AML, we injected it into the mice
   through the tail vein and found that after overexpressing Siglec6 in
   AML, the tumor’s proliferative ability and distribution level in the
   body were enhanced; whereas after knocking down Siglec6, the phenomenon
   was reversed, and the tumor distribution in the body was weakened,
   proving the importance of Siglec6 expression levels in the progression
   of AML. Next, we explored how Siglec6 promotes the progression of AML.
   Siglec6 is a transmembrane receptor that is specifically expressed on
   AML, and its cytoplasmic tail contains an ITIM and an ITIM-like motif,
   which have signal transduction functions. It can be phosphorylated by
   Src family kinases, thereby recruiting SH-2 domain-containing
   phosphatase SHP2, providing a molecular platform^[97]16. SHP2, full
   name Src homology 2 domain-containing protein tyrosine phosphatase, is
   a non-receptor type protein tyrosine phosphatase encoded by the PTPN11
   gene. SHP2 plays an important role in cellular signal transduction,
   especially in the invasion, metastasis, proliferation, apoptosis, and
   drug resistance of tumor cells. When SHP2 is activated, it promotes the
   phosphorylation of ERK^[98]17. ERK, full name extracellular
   signal-regulated kinase, is a member of the Mitogen-Activated Protein
   Kinases (MAPKs) family and is mainly involved in regulating key
   biological processes such as cell proliferation, differentiation,
   survival, and apoptosis^[99]18. When ERK is activated, it promotes the
   invasive migration and proliferation capabilities of tumors. At the
   same time, the overactivation of SHP2 protein also promotes the
   expression of IL-3 ^[100]19. IL-3, full name multi-colony stimulating
   factor, can act as a hematopoietic growth factor and activate
   cytokines, and its increased levels will promote the protein expression
   level of Siglec6, forming a positive feedback loop, further promoting
   the invasive migration and proliferation capabilities of AML^[101]20.
   Based on this, we set up in vitro experiments and chose MOLM-13 cells
   for analysis. We set up rescue experiments, dividing the cells into six
   groups: oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib,
   oe-Siglec6 + Dasatinib + Lovastatin, and
   oe-Siglec6 + Dasatinib + Lovastatin + IL-3 neutralizing antibody. We
   detected the protein expression levels of Siglec6, p-SHP2, IL-3,
   pERK1/2, proliferation-related proteins CyclinA1, CyclinD1, and
   invasion-related proteins MMP2 and MMP9 by Western blot. We found that
   compared with the oe-NC group, after overexpressing Siglec6, the
   protein expression levels of Siglec6, p-SHP2, IL-3, pERK1/2, CyclinA1,
   CyclinD1, MMP2, and MMP9 increased, and the corresponding use of Src
   inhibitors, SHP2 agonists, and IL-3 neutralizing antibodies reversed
   the protein expression levels. Transwell and CCK8 experiments also
   proved this point. It indicates that in AML, Siglec6 promotes the
   proliferation, invasion, and migration capabilities of AML through the
   SHP2/Src/ERK/IL-3 axis.

   Based on the previous research, we found that Siglec6 can affect the
   progression of AML through a series of molecular pathways, suggesting
   that Siglec6 can be a potential target for CAR-T. In this part, we
   focus on whether Siglec6 can be an effective target for CAR-T therapy.
   We modified T cells to target Siglec6 on AML. We co-cultured ordinary
   CD19 CAR-T, Siglec6 CAR-T with AML and detected the apoptosis level of
   AML cells by flow cytometry. We found that MOLM-13 cells cultured with
   Siglec6 CAR-T had stronger apoptosis capabilities. Further in vivo
   experiments were conducted by injecting MOLM-13 cells into NOD/SCID
   mice and treating the mice with CAR-T. In vivo imaging technology
   detected the distribution and content of tumors in the mice. We
   observed a significant phenomenon: the number of tumors in mice treated
   with Siglec6 CAR-T was significantly less than those treated with CD19
   CAR-T. This result indicates that Siglec6 CAR-T therapy may be more
   effective than CD19 CAR-T therapy in inhibiting tumor growth and
   reducing the number of tumors. For the occurrence of this phenomenon,
   we speculate that it may be due to the activated Siglec6 CAR-T cells
   producing interferons that directly attack tumor cells, leading to
   tumor cell death^[102]21. In related studies, it has been confirmed
   that CAR-T cells can kill tumor cells by releasing interferons and
   other cytokines^[103]22. Therefore, Siglec6 CAR-T cells may produce the
   killing effect on AML tumor cells by producing interferons and other
   cytokine.

   In summary, we have demonstrated that Siglec6 CAR-T can target Siglec6
   on AML and act on the SHP2/Src/IL-3/ERK axis to inhibit tumor
   progression. The discovery of this mechanism provides a molecular
   explanation for how Siglec6 CAR-T cells exert therapeutic effects on
   AML and offers potential intervention points for future treatment
   strategies. However, it is regrettable that we have not validated the
   mechanism of action of Siglec6 CAR-T at the clinical level. This
   limitation prompts us to further conduct clinical trials to evaluate
   the efficacy and safety of Siglec6 CAR-T cell therapy in actual
   patients, thereby providing more precise and effective treatment
   options for AML patients.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [104]Supplementary Material 1^ (97.3KB, pdf)
   [105]Supplementary Material 2^ (87KB, pdf)
   [106]Supplementary Material 3^ (12.2KB, docx)

Acknowledgements