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Imaging and detection of tumor-associated glycan structures on tumor cells

Periodic Reporting for period 2 - GlycoImaging (Imaging and detection of tumor-associated glycan structures on tumor cells)

Berichtszeitraum: 2019-04-01 bis 2021-09-30

A major challenge in the war against cancer is to find ways to diagnose and treat the disease at an early stage. Cancer occurs through a multistage process where cells are transformed to malignant tumors. Of essence is to discover the tumor at an early stage where the cancer is still curable. This calls for sensitive and effective diagnostic tools that can sense the cellular state early in the process.
In GlycoImaging we have addressed this need while exploiting novel molecularly imprinted polymers (MIPs) targeting glycans such as sialic acid, Tn and STn. These “plastic anti-bodies” were developed and used for visualization as cellular cancer biomarkers mainly in vitro using tumor cells cultured as monolayers and tumor cells cultured as 3D spheroids.
6 research groups, spread across 4 universities and institutes, and 1 industrial partner in 4 different countries have come together to train a new generation of chemists/biologists through the GlycoImaging PhD training network. Thereby we have created a highly interdisciplinary research-training network with ambitious scientific goals and an attractive training program.
GlycoImaging aimed to develop and implement glycan specific probes in clinically relevant cancer diagnostics technologies. To conclude, we have developed glycan-specific nano-probes in different core-shell formats as well as carbon nanodots (CNDs)—against sialic acid, Tn or STn, respectively, alongside digital holography for real-time visualization of cancer biomarkers and use in life science and education. Moreover, we have developed and analyzed novel fluorophores for detection of MIP binding, including guanidinium benzoxadiazole fluorescent probes. The MIPs have been modified for immune cell analyses by addition of PEG structures. By using the genetically engineered HEK293 CRISPR/Cas9 isogenic cell lines, cell libraries were generated of Tn and STn O-glycoforms as well as different mucins. Thereafter, secreted mucin constructs were used to identify and validate the glyco-structures in the library by mass spectrometry. Mucins such as MUC1, MUC2 and MUC7 have been analysed.
Summary of main results:

The protocols for the synthesis of fluorescent molecularly imprinted polymers (MIPs) of 100-200 nm size against the human sugar structure sialic acid (SA), SA-MIPs, have been developed, optimized and evaluated. The flow cytometry method show that SA-MIPs can target and bind to different cancer cells to a various degree, by detecting a fluorescent signal on the cancer cell. With fluorescence microscopy, the binding of the SA-MIPs to cancer cells was visualized. Specificity studies were performed by using pentavalent SA conjugates.

Flow cytometry and fluorescence microscopy also show that 20 nm red carbon nanodots (CNDs) and silica-coated red CNDs (R-CSNs) against the more cancer specific glycan biomarker sialylated Tn (STn) bind and target cancer cells. In addition, protocols for the synthesis of fluorescent 100 nm MIPs against human Tn, Tn-MIPs, were evaluated.

Digital holographic microscopy for measurements of cellular morphology in 3D, with or without MIPs, have been used analyze cancer cells. For that purpose, fluorescent or non-fluorescent SA-MIPs were evaluated. The SA-MIPs ability to affect cell motility and survival was investigated. The SA-MIPs seems to be non-inflammatory and non-toxic for phagocytosing cells in vitro. To determine the possibility to reduce possible unspecific uptake of SA-MIPs by phagocytosing cells, PEGylation of the SA-MIP particles was performed and tested. Indeed, the uptake of PEGylated MIPs was decreased.

Cell libraries were generated of Tn and STn O-glycoforms, as well as of different mucins by using genetically engineered HEK293 CRISPR/Cas9 isogenic cell lines. Thereafter, secreted mucin constructs were prepared for the use to identify and validate the glyco-structures in the library by mass spectrometry. Even more complex glycan structures on the mucins MUC1, MUC2 and MUC7 have been analysed. However, more work needs to be performed for using MIPs in binding assays with whole mucin constructs, since the specificity of the MIPs could not be verified with the current protocols. O-GalNAc truncation in cell lines such as MCF-7, MDAMB231 and PC3 cells was investigated for differences in cell motility and migration. Moreover, the energy metabolism profiles of O-GalNAc truncated PC3 cells were compared with PC3 WT cells.
Investigation of the ability of the glycosaminoglycan heparin to reduce cellular infection with SARS-CoV-2 was performed.
Preparations have been done in animal models to be able to study in vivo MIP-targeting to cancer cells in mice, using SA-MIPs with different fluorophores. More work needs to be performed to select the most optimal fluorophore for MIPs in future work.

A patent “Fluorescent particles with molecularly imprinted fluorescent polymer shells for cell staining applications in cytometry and microscopy” was filed in Germany including ESRs from GlycoImaging. Moreover, two additional patents were filed in 2020, not yet published or granted, both involving the work of ESRs in GlycoImaging.
GlycoImaging ESRs participated in fourteen different conferences presenting posters during 2019-2021. The conferences from April 2020 have been virtual. Seventeen publications with connection to GlycoImaging have been published between 2018-2021. Three doctoral theses´ were defended during 2021. The exhibition “Fighting cancer with plastic bullets” 2019-2020 at the Malmö University main library was a joint outreach activity together with EU-ITN Biocapture. The Graduate student symposium on Molecular Imprinting and a Summer School was co-organized with MU-ITN Biocapture in Berlin in August 2019 (BAM was the host). ESRs at BAM in Berlin participated in the public scientific event “Long night of Science” 2019.
The GlycoImaging project emphasized on highly needed sensitive, but low cost diagnostic tools and to bring together expertise in the field of cancer, biomarkers, glycobiology and imaging. The project made strongly impact on the researchers professional development in the preparations for a successful career, where the European industry also will make benefits.

High-quality ESR training with a commitment to train 9 ESRs toward a PhD degree was the central pillar of the project. The training had a focus on key scientific, business and entrepreneurial skills that are currently lacking in today´s European work force. The demand for researchers with this broad set of skills is widespread in both industry and academia.

At least nine manuscripts will be finalized with connection to GlycoImaging for publication in 2022 or later. Six more doctoral theses´ will be finalized and defended during 2021/2022.

The specific tasks in GlycoImaging included in the ESRs project plans, directly supported:

- specific targeting of rare cancer glycosylation motifs by the use of novel MIPs,
- enhanced understanding of the wide spectrum of alterations in glycosylation for the development and progression of neoplastic (cancer) diseases,
- the importance of research communication to schools and the society for better understanding of the needs in the research field
- improved knowledge of glycan-based experimental challenges and success rates,
- improvement of new cancer diagnostic tools worldwide,
- the innovation capacity by integrating molecular imprinting technology and assay development.
ESRs participating with posters in the "Biomarker" conference" in Malmö, October 2018
ESRs at BAM in Berlin participating in outreach activity Long night of Science 2019
Logo GlycoImaging
Four GlycoImaging ESRs playing the PhD game at the exhibition
Discussion between ESRs and PIs during meeting break at University of Copenhagen 2019
GlycoImaging Mid-term review meeting in Turku, May 2019
Researcher´s gallery exhibition - GlycoImaging ESRs visiting "Fighting cancer with plastic bullets"