. 2019 Aug;572(7769):392-396.

doi: 10.1038/s41586-019-1456-0. Epub 2019 Jul 31.

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy

Amira A Barkal^{1

2

3

4}, Rachel E Brewer^{1

2

3}, Maxim Markovic^{1

2

3}, Mark Kowarsky⁵, Sammy A Barkal¹, Balyn W Zaro^{1

2

3}, Venkatesh Krishnan⁶, Jason Hatakeyama^{1

7}, Oliver Dorigo⁶, Layla J Barkal⁸, Irving L Weissman^{9

10

11

12}

Affiliations

¹ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
² Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA, USA.
³ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
⁴ Stanford Medical Scientist Training Program, Stanford University, Stanford, CA, USA.
⁵ Department of Physics, Stanford University, Stanford, CA, USA.
⁶ Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Stanford University School of Medicine, Stanford, CA, USA.
⁷ Department of Urology, Stanford University School of Medicine, Stanford, CA, USA.
⁸ Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
⁹ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹⁰ Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹¹ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹² Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.

PMID: 31367043
PMCID: PMC6697206
DOI: 10.1038/s41586-019-1456-0

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy

Amira A Barkal et al. Nature. 2019 Aug.

. 2019 Aug;572(7769):392-396.

doi: 10.1038/s41586-019-1456-0. Epub 2019 Jul 31.

Authors

Affiliations

¹ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
² Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA, USA.
³ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA.
⁴ Stanford Medical Scientist Training Program, Stanford University, Stanford, CA, USA.
⁵ Department of Physics, Stanford University, Stanford, CA, USA.
⁶ Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Stanford University School of Medicine, Stanford, CA, USA.
⁷ Department of Urology, Stanford University School of Medicine, Stanford, CA, USA.
⁸ Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
⁹ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹⁰ Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹¹ Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.
¹² Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. irv@stanford.edu.

PMID: 31367043
PMCID: PMC6697206
DOI: 10.1038/s41586-019-1456-0

Abstract

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD47¹, programmed cell death ligand 1 (PD-L1)² and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)³. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers^4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

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Conflict of interest statement

Competing interests:

A.A.B. and I.L.W. are co-inventors on a patent application (62/684,407), which is related to this work. I.L.W. is a founder, director, stockholder, and consultant of FortySeven Inc., a cancer immunotherapy company.

Figures

**Extended Data Figure 1.**
Expression of innate immune checkpoints in human cancer a, Heatmap of expression (log₂(Normalized counts + 1)) of CD24 from bulk TCGA/TARGET studies, as compared to known innate immune checkpoint molecules, CD47, PD-L1, and B2M (tumor study abbreviations and n defined in Supplementary Table 1). b, Expression levels of CD24 in ovarian cancer (OV, red boxplot, n = 419) in comparison with ovarian tissue from healthy individuals (gray boxplot, n = 89), boxes show the median and whiskers indicate the 95^th and 5^th percentiles, ****P<0.0001, unpaired, two-tailed Student’s t-test. c, Expression levels of CD24 in TNBC (red boxplot, n = 124) in comparison with ER⁺PR⁺ breast cancer (ER⁺PR⁺, purple boxplot, n = 508) and normal breast (gray boxplot, n = 293). Each symbol represents an individual patient sample, boxes show the median and whiskers indicate the 95^th and 5^th percentiles, ****P<0.0001, one-way ANOVA with multiple comparisons correction, F_(2,922) = 95.80. d, Heatmap of marker gene expression (y-axis) across TNBC single cells (x-axis) and cell clusters identified (top). e, UMAP dimension 1 and 2 plots displaying all TNBC cells analyzed from six patients (n = 1001 single cells); cell clusters are colored by cell patient (key, left). f, CD24 vs. PD-L1 expression in the “Tumor epithelial cell” cluster for individual TNBC patients; number of single cells analyzed, PT039 n = 151 cells, PT058 n = 11 cells, PT081 n = 196 cells, PT084 n = 84 cells, PT089 n = 117, PT126 n = 60 cells. **P<0.01, ****P<0.0001. Data are violin plots showing median expression (log₂(Norm counts +1) and quartiles (paired, two-tailed t-test).

**Extended Data Figure 2.**
Flow-cytometry analysis of CD24 and Siglec-10 expression in human tumors and primary immune cells a, Gating strategy for CD24⁺ cancer cells and Siglec-10+ TAMs in primary human tumors; after debris and doublet removal, cancer cells were assessed as DAPI⁻CD14⁻ EpCAM⁺ and TAMs were assessed as DAPI⁻EpCAM⁻CD14⁺CD11b⁺. Plots are representative of 6 experimental replicates. b, (top) Representative flow cytometry histogram measuring the expression of Siglec-10 (blue shaded curves) versus isotype control (black lines) by non-cancerous peritoneal macrophages, numbers above bracketed line indicate percent macrophages positive for expression of Siglec-10; (bottom) frequency of peritoneal macrophages positive for Siglec-10 among all peritoneal macrophages as defined by isotype controls (n = 9 donors). c, Gating strategy for CD24⁺ cells and Siglec-10+ cells among PBMC cell types; after debris and doublet removal, monocytes were assessed as DAPI⁻CD3⁻CD14⁺; T cells were assessed as DAPI⁻CD14⁻CD3⁺; NK cells were assessed as DAPI⁻CD14⁻CD3⁻CD56⁺; B cells were assessed as DAPI⁻ CD56⁻CD14⁻CD3⁻CD19⁺. Plots are representative of 2 experimental replicates. d, Frequency of PBMC cell types positive for Siglec-10 (blue shaded bars) or CD24 (red shaded bars) out of total cell type (n = 3 donors). e, (left) Flow cytometry–based measurement of the surface expression of Siglec-10 on primary human donor-derived macrophages either unstimulated (top) or following stimulation with M2-polarizing cytokines TGFβ1 and IL-10 (bottom), numbers above bracketed line indicate percent CD11b+ macrophages positive for expression of Siglec-10; (right) Frequency of primary human donor-derived macrophages positive for Siglec-10 either without stimulation (Unstimulated, M0) or following stimulation with TGFβ1 and IL-10 (Stimulated, M2-like), (*n =* 30 Unstimulated donors, 33 Stimulated donors; unpaired, two-tailed Student’s t-test, ****P<0.0001, data are mean ±s.e.m.). f, Flow cytometry–based measurement of phagocytosis of MCF-7 cells by unstimulated donor-derived macrophages (white dots) versus TGFβ−1 and IL-10-stimulated donor-derived macrophages (n = 3 donors, unpaired, one-tailed t-test, *P = 0.0168). g, (left) Flow cytometry–based measurement of the surface expression of Siglec-10 on matched, primary donor-derived macrophages either unstimulated (gray shaded curve), or following stimulation with TGFβ1 and IL-10 (blue line), or IL-4 (green line); (right) Frequency of matched, human donor-derived macrophages positive for Siglec-10 either without stimulation (Unstimulated, M0), or following stimulation with TGFβ1 and IL-10 (blue dots), or stimulated with IL-4 (n = 4 donors). Data are mean ±s.e.m.

**Extended Data Figure 3.**
Siglec-10 binds to CD24 expressed on MCF-7 cells a, Flow cytometry histogram measuring binding of Siglec-10 to WT MCF-7 cells (blue shaded curve) versus ΔCD24 MCF-7 cells (red shaded curve). Data are representative of two experimental replicates. b, Merged flow cytometry histogram measuring binding of Siglec-10-Fc to WT MCF-7 cells treated with heat-inactivated neuraminidase (WT-HI NA, blue line), WT MCF-7 cells treated with neuraminidase (WT-NA, green line), ΔCD24 MCF-7 cells treated with heat-inactivated neuraminidase (red line, ΔCD24-HI NA), and ΔCD24 MCF-7 cells treated with neuraminidase (purple line, ΔCD24-NA) as compared to isotype control (black line). Data are representative of two experimental replicates. c, Flow cytometry–based measurement of phagocytosis of CD24⁺ parental MCF-7 cells (WT) and CD24⁻ (ΔCD24) MCF-7 cells by co-cultured human macrophages in the presence of neuraminidase (+NA) or heat-inactivated neuraminidase (+HI-NA) (n = 4 donors; two-way ANOVA with multiple comparison’s correction, cell line F_(1,12) = 180.5, treatment F_(1,12) = 71.12, ****P<0.0001, data are mean ±s.e.m.). **f,h** Representative flow cytometry histogram measuring the binding of Siglec-5, f, or Siglec-9, h, to WT MCF-7 cells treated with either vehicle (blue shaded curve) or neuraminidase (green shaded curve). Data are representative of two experimental replicates. **g,i,** Frequency of macrophages positive for Siglec-5, g, or Siglec-9, i, among unstimulated M0 macrophages or stimulated M2-like macrophages (n = 4 donors). Data are mean ±s.e.m.

**Extended Data Figure 4.**
Anti-CD24 monoclonal antibodies promote phagocytic clearance of cancer cells over time a, Schematic of CD24-Siglec-10 inhibition of phagocytosis; the inhibitory receptor Siglec-10 engages its ligand CD24 on cancer cells, leading to phosphorylation of the two ITIM motifs in the cytoplasmic domain of Siglec-10 and subsequent anti-inflammatory, anti-phagocytic signaling cascades mediated by SHP-1 and SHP-2 phosphatases; upon the addition of a CD24 blocking antibody, macrophages are disinhibited and thus capable of phagocytosis-mediated tumor clearance. b, Quantification of phagocytosis events (red⁺) of MCF-7 cells treated with anti-CD24 mAb (red curve) versus IgG control (blue curve) as measured by live-cell microscopy over time in hours (h), normalized to maximum measured phagocytosis events per donor, (n = two donors; P value computed by two-way ANOVA of biological replicates, F_(1,24) = 65.02). Line is the mean of two biological replicates with individual replicates shown. c, Representative fluorescence microscopy images of *in vitro* phagocytosis of MCF-7 cells (mCherry⁺, red) by macrophages (Calcein, AM; green) in the presence of IgG control (left), anti-CD24 mAb (middle), or anti-CD24 mAb and anti-CD47 mAb (right), after 6 hours of co-culture. Experiment was repeated with three donors. Scale bar represents 100 μm. d, Representative Z-stack images collected from high-resolution confocal fluorescence microscopy of macrophage phagocytosis demonstrating engulfment of whole MCF-7 cells (mCherry⁺, red) by macrophages (Calcein, AM; green). Experiment was repeated with three donors. Scale bar represents 50 μm.

**Extended Data Figure 5.**
CD24 antibody blockade of CD24-Siglec-10 signaling promotes dose-responsive enhancement of phagocytosis a, Gating strategy for *in vitro* phagocytosis assay. Following debris and doublet removal, phagocytosis was assessed as the frequency of DAPI⁻CD11b⁺FITC⁺ events out of all DAPI⁻CD11b⁺ events. Numbers indicate frequency of events out of previous gate. Plots are representative of at least 10 experimental replicates. b, Dose-response relationship of anti-CD24 mAb on phagocytosis of MCF-7 cells, concentrations listed on the x-axis as compared to IgG control (n = 3 donors). Connecting line is mean. c, Flow cytometry–based measurement of phagocytosis of NCI-H82 cells by donor-derived macrophages (n = 3 donors) in the presence of anti-CD24 mAb as compared to IgG control; each symbol represents an individual donor (paired, two-tailed Student’s t-test, ****P =* 0.0001). Data are mean ±s.e.m. d, Flow cytometry–based measurement of phagocytosis of CD24⁺ parental MCF-7 cells (WT) and CD47⁻ (ΔCD47) MCF-7 cells by co-cultured human macrophages, in the presence or absence of anti-CD24 mAb (horizontal axis), (n = 4 donors; two-way ANOVA with multiple comparisons correction, cell line F_(1,8) = 6.490; treatment F_(1,8) = 98.73, **P = 0.0054). Data are mean ±s.e.m. e, Flow cytometry –based measurement of phagocytosis of Panc1 pancreatic adenocarcinoma cells in the presence of anti-CD24 mAb, cetuximab (anti-EGFR), or both anti-CD24 mAb and cetuximab, as compared to IgG control (n = 6 donors) (one-way ANOVA with multiple comparisons correction, F_(3,20) = 66.10. *P = 0.0373, **P = 0.0057. Data are mean ±s.e.m.

**Extended Data Figure 6.**
The opsonization effect of anti-CD24 mAb is minor and CD24 blockade promotes phagocytosis of primary TNBC a, (left) Representative flow cytometry histogram measuring the expression of EpCAM (green shaded curve) by parental MCF-7 cells, number above bracketed line indicates percent MCF-7 cells positive for expression of EpCAM; (right) Flow cytometry–based measurement of phagocytosis of parental MCF-7 cells by co-cultured human macrophages, in the presence of either IgG control, anti-EpCAM mAb, or anti-CD24 mAb (n = 4 donors; repeated measures ANOVA with multiple comparisons correction, F_(2,9) = 340.9, *P = 0.0287, **P = 0.0015, ****P<0.0001). Data are mean ±s.e.m. b, Fold change in phagocytosis by M0 (unstimulated) or M2-like (TGFβ−1, IL-10-stimulated) macrophages upon the addition of anti-EpCAM mAb as compared to IgG control, (n = 9 donors. Paired, two-tailed t-test, NS = not significant). Data are mean ±s.e.m. c, Flow cytometry–based measurement of anti-CD24 mAb-induced phagocytosis of MCF-7 cells by untreated macrophages (white bar) versus macrophages treated with anti-CD16/32 mAb (+FcR blockade, blue bar) (n = 3 macrophage donors. Paired, two-tailed t-test. Each point represents an individual donor. *P = 0.0358). Data are mean ±s.e.m. d, Response to anti-CD24 mAb by M2-like macrophages vs. M0 macrophages; each symbol represents an individual donor (*n =* 4 M0 donors, n = 6 M2-like donors; unpaired, two-tailed Student’s t-test, *P = 0.0160). e, Pearson correlation between stimulated (M2-like) donor-derived macrophage Siglec-10 expression (MFI = median fluorescence intensity) (x-axis) and response to anti-CD24 mAb as computed by the phagocytosis fold change between anti-CD24 mAb treatment and IgG control (y-axis), (n = 7 donors); exponential growth curve is shown. f, Spearman correlation between cancer cell CD24 expression (MFI = median fluorescence intensity) (x-axis) and baseline, un-normalized phagocytosis levels (IgG control) averaged across all donors per cell line. Exponential growth equation is shown. (n are same as in Figure 3b and Extended Data Figure 5c, *P = 0.0417). Data are mean ±s.e.m. g, Flow cytometry–based measurement of phagocytosis of a patient sample of primary TNBC cells in the presence of anti-CD24 mAb, anti-CD47 mAb, or both anti-CD24 mAb and anti-CD47 mAb, as compared to IgG control (n = 3 macrophage donors challenged with n = 1 primary TNBC donor. Repeated measures one-way ANOVA with multiple comparisons correction, F_{(1.217,2.434)} = 26.17). Each point represents an individual donor. NS = not significant, *P = 0.0434, **P = 0.0028. Data are mean ±s.e.m.

**Extended Data Figure 7.**
Gating strategy for *in vivo* phagocytosis Gating strategy for *in vivo* TAM phagocytosis of MCF-7 cells; following debris and doublet removal, TAM phagocytosis assessed as the frequency of DAPI⁻CD11b⁺F4/80⁺GFP⁺ events out of total DAPI⁻CD11b⁺F4/80⁺ events; M1-like TAMs assessed as DAPI⁻CD11b⁺F4/80⁺CD80⁺, Numbers indicate frequency of events out of previous gate. Plots are representative of three experimental replicates.

**Extended Data Figure 8.**
Characterization of MCF-7 WT and MCF-7ΔCD24 cells *in vitro* and *in vivo* a, Representative flow cytometry plots demonstrating TAM phagocytosis in GFP-luciferase⁺ CD24⁺ (WT) MCF-7 tumors (left) vs. CD24⁻ (ΔCD24) MCF-7 tumors (middle), numbers indicate frequency of phagocytosis events out of all TAMs; (right) frequency of phagocytosis events out of all TAMs in WT tumors vs. ΔCD24 tumors 28 days after engraftment (WT n = 10, ΔCD24 n = 9. Unpaired, two-tailed Student’s t-test, ****P<0.0001). b, Frequency of TAMs positive for CD80 (M1-like) as per gating in a, among all TAMs macrophages as defined by fluorescence minus one controls (WT n = 10, ΔCD24 n = 9. Unpaired, two-tailed Student’s t-test, *P<0.0203). Data are mean ±s.e.m. c, *In vitro* proliferation rates of MCF-7 WT and MCF-7ΔCD24 as assessed by confluence percentage (y-axis) over time (x-axis), (n = 6 technical replicates, one experimental replicate) Individual technical replicates shown, connecting line indicates mean. d, Flow cytometry-based measurement of the surface expression of CD24 on MCF-7 cells (blue shaded curve) versus CD24 knockout cells (ΔCD24) (red shaded curve) prior to tumor engraftment as compared to isotype control (black line), numbers above bracketed line indicate percent MCF-7 WT cells positive for expression of CD24. Plot is representative of 10 experimental replicates. e, (left) Representative flow cytometry histogram of the surface expression of CD24 on Day 35 WT MCF-7 tumors (blue shaded curve) versus Day 35 CD24 knockout tumors (ΔCD24) (red shaded curve) as compared to isotype control (black line); (right) flow cytometry–based measurement of the frequency of CD24⁺ cells among all cancer cells in Day 35 WT tumors versus Day 35 ΔCD24 tumors (WT n = 4, ΔCD24 n = 4). Data are mean ±s.e.m. f, Representative flow cytometry plots of tissue-resident macrophages out of total live cells in vehicle-treated animals (left) vs. anti-CSF1R-treated animals (middle), numbers indicate frequency of CD11b+,F4/80+ macrophage events out of total live events; (right) frequency of TAMs (CD11b+,F4/80+) out of total live cells in vehicle-treated animals (n = 5, blue shaded boxplot) vs. anti-CSF1R-treated animals (n = 4, red shaded boxplot) as measured by flow cytometry. **P<0.01. Boxplots depict mean and range.

**Extended Data Figure 9.**
Validation of CD24 inhibition in *in vivo* models of ovarian and breast cancer a, *In vivo* phagocytosis of WT or ΔCd24a cancer cells by mouse TAMs Flow cytometry–based measurement of *in vivo* phagocytosis of CD24⁺GFP⁺ ID8 cells (WT) versus CD24⁻GFP⁺ ID8 cells (ΔCd24a) by mouse peritoneal macrophages, (*n =* 5 mice; unpaired, two-tailed Student’s t-test with multiple comparisons correction, *P = 0.0196). b, Representative bioluminescence image of tumor burden in C57Bl/6 mice with ID8 WT vs. ID8ΔCd24a tumors (image taken 49 days post-engraftment and representative of one experimental replicate). c, Burden of ID8 WT tumors (blue) vs. ID8ΔCd24a tumors (red) as measured by bioluminescence imaging (WT n = 5, ΔCd24a n = 5. Two-way ANOVA with multiple comparisons correction, tumor genotype F_(1,48) = 10.70, ***P = 0.0001). Data are representative of one experimental replicate. d, Extended measurement (as in Figure 4e) of burden of MCF-7 WT tumors treated with IgG control (blue) vs. anti-CD24 mAb (red) as measured by bioluminescence (IgG control n = 5, anti-CD24 mAb *n =* 5. Days on which anti-CD24 mAb was administered are indicated by arrows below x-axis. Data are of one independent experimental cohort. Two-way ANOVA with multiple comparisons correction, tumor treatment F_(1,81) = 16.75). ****P<0.0001. Data are mean ±s.e.m.

**Extended Data Figure 10.**
Anti-CD24 mAb induces B cell clearance but does not bind human RBCs, and CD47 and CD24 subset human DLBCL demonstrating inversely correlated expression a, Flow cytometry–based measurement of phagocytosis of B cells (n = 4 donors, pooled) by donor-derived macrophages (n = 4 donors) in the presence of anti-CD24 mAb as compared to IgG control; each symbol represents an individual donor (paired, two-tailed Student’s t-test, ***P = 0.0008). b, (left) Representative flow cytometry histogram measuring the expression of CD24 (red line) and CD47 (blue line) by human RBCs; (right) Flow-cytometry–based measurement of the frequency of CD24⁺ versus CD47⁺ RBCs out of total RBCs (n = 3 donors). Data are mean ±s.e.m. c, (left) Expression levels in log₂(norm counts + 1) of CD24 and CD47 in Diffuse Large B Cell Lymphomas from TCGA (n = 48), data are divided into quadrants by median expression of each gene as indicated by dotted lines, number and percentage of total patients in each quadrant indicated on plot. Each dot indicates a single patient; (right) 2-dimensional contour plot of Diffuse Large B Cell Lymphoma patients in left plot.

**Figure 1.**
CD24 is over-expressed by human cancers and is co-expressed with Siglec-10 on TAMs a, Heatmap of CD24 tumor to matched normal expression ratios (log₂FC) compared to known immune checkpoints (tumor study abbreviations and n defined in Supplementary Table 1). **b,c,** Relapse-free survival percentage (RFS) for ovarian cancer patients (n = 31), b, and overall survival percentage (OS) for breast cancer patients (n = 1080), c, with high versus low CD24 expression as defined by median. Two-sided P value computed by a log-rank (Mantel-Cox) test. Numbers of subjects at risk in high group (red) vs. low group (blue) indicated below the x-axes. d, UMAP dimension 1 and 2 plots displaying TNBC cells from 6 patients (n = 1001 single cells); (left) cells colored by cluster identity, (right) CD24 (red) and Siglec-10 (blue) expression overlaid onto UMAP space as compared to CD47 (gray) and PD-L1 expression (gray). e, (left) Representative flow cytometry histogram of CD24 expression by ovarian cancer (OV) cells (top) or breast cancer (BRCA) cells (bottom); (right) frequency of CD24⁺ cancer cells in ovarian cancer (n = 3 donors) (top) or breast cancer (n = 5 donors) (bottom). Data are mean ±s.e.m. f, (left) Representative flow cytometry histogram measuring the expression of Siglec-10 by ovarian cancer (OV) TAMs (top) or breast cancer (BRCA) TAMs (bottom); (right) frequency of Siglec-10⁺ TAMs in ovarian cancer (n = 6 donors) (top) or breast cancer (n = 5 donors) (bottom). Data are mean ±s.e.m.

**Figure 2.**
CD24 directly protects cancer cells from phagocytosis by macrophages a, Schematic depicting interactions between macrophage-expressed Siglec-10 and CD24 expressed by cancer cells. b, Phagocytosis of CD24⁺ MCF-7 cells (WT) and CD24⁻ (ΔCD24) MCF-7 cells, in the presence or absence of anti-CD47 mAb, (n = 4 donors; two-way ANOVA with multiple comparisons correction, cell line F_(1,12) = 65.65; treatment F_(1,12) = 40.30, **P = 0.0045, ****P<0.0001). c, Representative phagocytosis images of pHrodo-red⁺, GFP⁺ MCF-7 cells (WT, top; ΔCD24, bottom) over time (hours); images representative of two donors. d, Phagocytosis of WT MCF-7 cells, in the presence of anti-Siglec-10 mAb or IgG control (n = 4 donors; paired, two-tailed Student’s t-test, ***P = 0.0010). e, (left) FACS–based measurement of Siglec-10 expression by Siglec-10 KO macrophages (red) vs. Cas9 control (blue); (right) Frequency of Siglec-10⁺ macrophages among Cas9 control vs. Siglec-10 KO macrophages. Data are mean±s.e.m of n = 5 donors. f, Phagocytosis of WT MCF-7 cells by either Siglec-10 KO or Cas9 control macrophages. Data are mean±s.e.m of n = 5 donors; paired, one-tailed Student’s t-test, **P = 0.0035. g, Flow cytometry–based measurement of binding of recombinant Siglec-10-Fc to MCF-7 WT cells treated with neuraminidase (+NA) or heat-inactived neuraminidase (+HI-NA); plot is representative of two experimental replicates. h, (left) Flow cytometry–based measurement of binding of Siglec-10-Fc to neuraminidase-treated MCF-7 WT cells vs. neuraminidase-treated MCF-7ΔCD24 cells. Plot is representative of 3 experimental replicates; (right) normalized binding of Siglec-10-Fc to neuraminidase-treated MCF7ΔCD24 cells vs. neuraminidase-treated MCF7 WT cells. Data are representative of 3 experimental replicates. i, Representative images from live-cell microscopy phagocytosis assays of pHrodo-red⁺ MCF-7 cells treated with anti-CD24 mAb (right) or IgG control (left) at t = 5:05 h; images are representative of two donors and two experimental replicates.

**Figure 3.**
Treatment with anti-CD24 mAb promotes phagocytic clearance of human cancer cells a, Representative flow cytometry plots depicting phagocytosis of MCF-7 cells treated with anti-CD24 mAb, CD47 mAb, or dual treatment vs. IgG control. Plots representative of 5 donors. b, Phagocytosis of MCF-7 (n = 5 donors), APL1 (n = 8 donors), and Panc1 (n = 8 donors) (left) and U87-GM cell line (n = 3 donors; solid bars) (right) in the presence of anti-CD24 mAb, anti-CD47 mAb or dual treatment vs. IgG control (one-way ANOVA with multiple comparisons correction; MCF-7 F_(3,16) = 145.6, APL1 F_(3,28) = 144.7, Panc1 F_(3,28) = 220.7, U-87 MG F_(3,8) = 200.4; NS = not significant, **P = 0.0092, ***P = 0.0001, ****P<0.0001). c, Response to anti-CD24 mAb by Siglec-10 KO vs. Cas9 control macrophages (n = 4 donors, connecting lines indicate matched donor. Paired, one-tailed Student’s t-test, **P = 0.0089) d, Pearson correlation between CD24 expression (x-axis) and mean anti-CD24 mAb response (y-axis) (n are same as listed in b, and Extended Data Figure 5c. Linear regression is shown. Error bars are mean ±s.e.m. *P = 0.0375). e, Workflow to measure phagocytosis of primary ovarian cancer, f, Phagocytosis of primary ovarian cancer cells treated with anti-CD24 mAb, anti-CD47 mAb, or dual treatment vs. IgG control (n = 10 macrophage donors, n = 1 primary ovarian cancer ascites donor) (one-way ANOVA with multiple comparisons correction, F_{(2.110, 18.99)} = 121.5, **P = 0.0078, ***P = 0.0006, ****P<0.0001). Data are mean ±s.e.m.

**Figure 4.**
CD24 protects cancer cells from macrophage attack *in vivo* a, Representative bioluminescence image of Day 21 tumors in mice engrafted with MCF-7 WT vs. MCF-7ΔCD24 tumors (image representative of two independent experimental cohorts). b, Burden of MCF-7 WT vs. MCF-7ΔCD24 tumors in mice with TAMs (vehicle) or TAM-depleted mice (anti-CSF1R) as measured by bioluminescence (WT vehicle n = 14, WT TAM depletion n = 5, ΔCD24 vehicle n = 13, ΔCD24 TAM depletion n = 5. Two-way ANOVA with multiple comparisons correction, tumor genotype F_(3,33) = 11.75, *P = 0.0296, ***P = 0.0009). c, Survival analysis of vehicle-treated mice in c, P value computed by a log-rank (Mantel-Cox) test (WT n = 5, ΔCD24 n = 5). d, Representative bioluminescence image of Day 33 tumors in mice with MCF-7 tumors treated with either IgG control or anti-CD24 mAb (image representative of two experimental cohorts). Data are mean ±s.e.m. e, Burden of MCF-7 WT tumors treated with IgG control (blue) vs. anti-CD24 mAb (red) as measured by bioluminescence (IgG n = 10, anti-CD24 mAb *n =* 10. Days of anti-CD24 mAb administration indicated by arrows. Data of two experimental cohorts. Two-way ANOVA with multiple comparisons correction, tumor treatment F_(1,126) = 5.679, ****P <0.0001).

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Comment in

CD24 - a novel 'don't eat me' signal.
Bradley CA. Bradley CA. Nat Rev Cancer. 2019 Oct;19(10):541. doi: 10.1038/s41568-019-0193-x. Nat Rev Cancer. 2019. PMID: 31406301 No abstract available.
CD24 - a novel 'don't eat me' signal.
Bradley CA. Bradley CA. Nat Rev Drug Discov. 2019 Sep;18(10):747. doi: 10.1038/d41573-019-00146-0. Nat Rev Drug Discov. 2019. PMID: 31570841 No abstract available.
Innate immune checkpoints for cancer immunotherapy: expanding the scope of non T cell targets.
Campesato LF, Weng CH, Merghoub T. Campesato LF, et al. Ann Transl Med. 2020 Aug;8(16):1031. doi: 10.21037/atm-20-1816. Ann Transl Med. 2020. PMID: 32953831 Free PMC article. No abstract available.

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CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy

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CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy

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