citations

                    Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions
                

Authors:

Boelaars K, Rodriguez E, Huinen ZR, Liu C, Wang D, Springer BO, Olesek K, Goossens-Kruijssen L, van Ee T, Lindijer D, Tak W, de Haas A, Wehry L, Nugteren-Boogaard JP, Mikula A, de Winde CM, Mebius RE, Tuveson DA, Giovannetti E, Bijlsma MF, Wuhrer M, van Vliet SJ, van Kooyk Y

In:

Source: Nat Commun.
Publication Date: (2024)
Issue: 7(1): 430

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Gene Expression

Basic Research

Molecular Biology

Drug Discovery

Cells used in publication:

Monocyte, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

CRISPR-cas9 gene knockout
Generation of a Siglec-9 knockout (KO) in monocytes was done using nucleofection of freshly isolated CD14+ monocytes, as reported previously 77. First, 6 µL of 160 µM crRNA (Dharmacon, CM-012842-01-0010) and 6 µL of 160 µM tracrRNA (Dharmacon, U-002005-50) were mixed and incubatedat 37 °C to form 12 µLof gRNA:tracrRNA duplex specific for Siglec-9. Subsequently, 12 µL of 40 µM Cas9-NLS protein (Horizon Discoveries, CAS12206) was added, after which the sample was incubated for 15 minutes at 37 °C to form CRISPR-Cas9 ribonucleoproteins (crRNP). The crRNPs were stored at –70 °C until use. Freshly isolated CD14+ monocytes were nucleofected with crRNP using the P3 Primary Cell 4D-Nucleofector TM X Kit L (Lonza, V4XP-3034) according to the manufacturer’s protocol. The mock transfection was performed by exposure to the nucleofection process without crRNPs present. Per condition, 12.5 µL crRNP and 5 × 10^6 CD14+ monocytes in 100 µL P3 Primary Cell NucleofectorTM solution were added to the NucleovetteTM vessel. After nucleofection (pulse code DK-100), cells were resuspended in pre-warmed complete RPMI and incubated for 30 minutes at 37 °C. After incubation, cells were harvested from the Nucleovette TM vessel, counted, and used for co-culture experiments.

Abstract

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be
overexpressed by cancer cells. Here,we unveil a previously unrecognized role of sialic acid-containing glycans onPDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

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