Cancer's sweet spot: techniques to harness saccharides in tumor biology

Why are tumors sickly sweet?

As interest built in the molecular aspects of cancerous cells, pathologists who studied the intricate details of tumors noticed that cancerous cells have a markedly higher amount of a simple sugar, termed sialic acid, on their cell surface compared to healthy cells. Hypersialylation has been observed in several different cancer types including lung, hematologic, breast and pancreatic tumors [1,2].

Exploring why tumor cells have evolved to have more glycans, it was discovered that sialylation (the process of adding a sialic acid unit to the end of an oligosaccharide chain of a glycoprotein) enhances the tumor cell's motility, allowing it to invade and metastasize more effectively, whilst also protecting the tumor from cell death induced by chemotherapeutic drugs, galectins and death receptor ligands [3].

Exploiting cancer's sweet spot

In 2018, James Allison (MD Anderson Cancer Center, TX, USA) and Tasuku Honjo (Kyoto University, Japan) were awarded the Nobel Prize in Physiology & Medicine 2018 for their research on exploiting the inhibition of negative immune regulation as a therapeutic strategy [4]. The duo uncovered PD-1 and CTLA-4 as key targets that could achieve complete remission for some patients [5].

Cure and cancer are words that prior to this, were never heard together. Yet the work of Honjo and Allison paved the way to change the prognosis of some cancer patients from 6–9 months to live, to being free of their cancer indefinitely [6].

However, these so-called miracle drugs don't work for most patients, even in indications such as melanoma, for which immuno-oncology is most effective [7]. With a large percentage of patients being unresponsive to immunotherapy, a big question remained. Are there other pathways that tumors use to evade the immune system?

This brings us to the deceitfully simple sugar: sialic acid. Fast forward a few years and in 2022 the Nobel Prize in Chemistry was part-awarded to Carolyn Bertozzi (Stanford University, CA, USA) for advancing click chemistry and biorthogonal chemistry, two groundbreaking techniques that are enhancing the effectiveness of cancer treatments [8].

Siglec: a piece of the puzzle ‘clicks’ into place

The ingenuity of creative chemists has made it possible to make almost any biological structure imaginable. However, while possible, this assembly is by no means a simple feat. The intricate process of forming linkages between molecules, managing a myriad of possible variables and maintaining highly optimized conditions made molecular synthesis a cumbersome endeavor.

In the late 1990s, K Barry Sharpless (Scripps Research, CA, USA) began devising a streamlined approach for manufacturing compounds, using a limited set of reactions, kickstarting the concept of click chemistry. There were strict rules regarding which reactions made the mark; each reaction should operate under simple conditions; generate only inoffensive by-products and be easily isolated by non-chromatographic methods such as crystallization [9,10]. By standardizing the process, Sharpless not only made manufacturing processes more efficient but also democratized the field of click chemistry, making it accessible to the wider research community.

The linkages during click chemistry work on the premise of attaching small chemical buckles to a wide array of different biomolecules, allowing for molecules to be ‘snapped’ together, in a similar way to LEGO pieces being joined to create structures of greater complexity. This set of powerfully selective ‘blocks’ provides researchers with an IKEA-like set of materials and instructions that work reliably in both small- and large-scale applications [9].

The reaction that has become most synonymous with click chemistry is 1,3-dipolar cycloadditions. This highly effective reaction is widely used across the pharmaceutical industry. Sharpless and Morten Meldal (University of Copenhagen, Denmark) discovered, independently of each other, that copper is a remarkable catalyst for this process, cementing it as an elegant and immensely effective method for creating complex structures [8].

Bertozzi's work has built on that of her fellow Laureates, in transforming the process from a copper-based system to a sialic acid foundation, allowing conjugational chemistry to take place within living systems and opening a floodgate for research opportunities. The overarching process of carrying out click chemistry in biological environments has been coined ‘bioorthogonal chemistry’ [11].

The key benefit of using a sialic acid-based system for treating cancer is tied to the widespread presence of sugar on immune cells as it has been demonstrated that checkpoint inhibitor drugs are less effective in cancer patients with a high density of sialic acid on their malignant cells [12]. At the touchpoint between the immune system and cancerous cells lies a collection of sensors including immune checkpoint receptors such as the aforementioned PD-1 and CTLA-4. The game changer in Bertozzi's work hinges on the fact that the receptor for sialic acid – Siglec – is present not just on T cells (where CTLA-4 is found), but on almost every type of immune cell in the human body [12]. This includes dendritic cells, NTK cells and macrophages: all cells that are all vital in eliciting and implementing an immune response to cancer cells.

There are 15 different Siglec (sialic acid-binding immunoglobulin-like lectin) receptors on the surface of human cells, some of which have a very similar composition to the cytosolic section of PD-1 [12,13], indicating that they could have an immunosuppressive effect. This led Bertozzi's team to hypothesize that sialic acid could be utilized as an immuno-therapeutic target, in a similar way to PD-1 and CTLA-4.

To test this hypothesis, researchers have worked on developing a compound that uses click chemistry to combine several molecules and elicit a more effective antitumor-immune response. This compound comprises four elements; a monoclonal antibody to target cancerous cells, a checkpoint inhibitor, a sialidase enzyme to cleave the sugars and a biotin molecule to allow the researchers to gain a visual perspective of how successfully the construct has performed. The compound is designed to potently and selectively remove sialoglycans on the surface of cancer cells, reducing the activation of Siglec receptors and optimizing the performance of the checkpoint inhibitor [12–14].

Click chemistry has expedited this process, allowing the team to test lots of combinations of click ‘handles’ to work out which may work best in vivo. Through animal model studies, teams have demonstrated that removing sialic acid from the surface of cancer cells repolarizes tumor-associated macrophages thereby improving immune checkpoint blockade and delaying tumor growth [15–17]. Expanding upon insights garnered from animal model studies, this work has transitioned into clinical trials. A Phase I trial of Bertozzi's glycol-immune checkpoint inhibitor (called E-602) has solidified its position as a promising candidate; E-602 has exhibited evidence of both dose-dependent desialylation and immune system activation [17,18].

Studies presented at the Society for Immunotherapy of Cancer's (SITC) 38th Annual Meeting (1–5 November 2023, CA, USA) have further highlighted how other research teams are looking to make use of different aspects of glycobiology to enhance immuno-oncology. Bashian et al. demonstrated that the knockout of a single glycosyltransferase in adoptively transferred T cells improves tumor control and survival in murine models of melanoma [19]. Zhang et al., meanwhile, showed that β-Glycans can be used to clear treatment-resistant ovarian cancer cells from the peritoneal fluid, unveiling a potential new method to control their metastasis [20].

Until recently the power of glycans to predict a patient's response to immunotherapy has been hampered by a lack of standardization of glycol-analytical tools. Alternative standalone biomarkers including PD-L1, tumor mutational burden and microsatellite instability have exhibited limited efficacy when studied in silos. Therefore, there is now a great effort to develop platforms that can conduct glycoproteomic profiling. Several methods are involved in identifying prognostic glycogenes, including Univariate Cox, LASSO regression, Multivariate Cox analyses and Kaplan-Meier methods. In melanoma, breast and colorectal cancer, researchers have reported success in predicting immunotherapy outcomes by creating glycol-immune signatures [21,22].

The evolution of techniques to analyze & quantify sialic acid

Technological advancements have demonstrated just how big a role sugar plays in cancer biology, but how can accurate measurements be taken to help stratify patients for therapy based on their sugar profiles?

The journey to effectively detect and quantify sialic acid begins with colorimetric assays. Using mild acid hydrolysis, researchers worked on breaking bonds between sialic acid and its protein and lipid attachments. Featuring the likes of Orcinol, Resorcinol and Thiobarbiturate; colorimetric assays have suffered from poor specificity and sensitivity, leading scientists to search for more sensitive methods [23].

Enter the fluorometric assays, whereby an oxidant or acid reacts with sialic acid to produce a dazzling fluorophore, yielding a highly sensitive measurement, although their specificity leaves something to be desired. Like colorimetric methods, they are easy to perform with basic laboratory equipment, but they differ in that samples are cumbersomely long to process – not ideal when a lab has a large number of samples to work through.

In response to the limitation of specificity, researchers worked on developing a more specific technique, leading to enzymatic methods. Enzymatic methods are favorable in that they can be combined with the aforementioned assays and due to the specificity of enzymes, this method is exceptionally precise at identifying sialic acids. However, once again the challenge of time crops up – enzymatic assays require numerous steps and lengthy reaction times, plus the enzymes themselves are expensive.

Building on previous methods, chromatography has gained great momentum due to its high specificity and its ability to separate, identify and quantify sialic acids in biological samples. Early chromatographic methods included gas chromatography (GC) and high-performance liquid chromatography; these preliminary methods lacked sensitivity levels needed and were only capable of quantifying large, standardized samples. The advancement of chromatography has come from combining it with other methods such as colorimetric assays or florescent tagging systems.

Next in steps in mass spectrometry (MS). First, MS was combined with chromatography in the form of GC-MS, which boasts a much greater sensitivity than aforementioned methods. Fast forward, MS and its derivatives have emerged as an effective way to identify aberrant glycosylation from target serum glycoproteins or global serum/plasma/tissue proteins [24,25]. The biggest advantage of using MS is the detailed structural information that can be gained, which is important when detecting subtle changes in glycan structures.

Liquid chromatography mass spectrometry (LC-MS) has sealed itself as the ‘go-to’ technique for small molecule bioanalysis due to its favorable selectivity, sensitivity, and robustness [26]. Samples must be prepared for LC-MS analysis by releasing the glycans from their protein partner, either using an enzyme or via a chemical release process such as hydrazinolysis. The released glycans then need to be derived into more stable compounds by permethylation. Once permethylated the glycans should then be fluorescently labeled to aid high-resolution analysis [27].

Traditionally viewed solely as a method for separating oligosaccharides within the MS process, capillary electrophoresis (CE) has come to the fore as a powerful analytical technique within its own right for characterizing carbohydrates [28]. When profiling small quantities of clinical samples for glycans, MALDI-MS has emerged as a favorable approach. Some advantages of MALDI-MS include the technique's quick and relatively easy processes that allow for the detection of a large mass range. However, it is limited in its ability to determine structural isomers' different branching patterns and linkage positions [29].

The evolution of these analytical techniques combined with the chemical engineering method of click chemistry is ushering in a new era of immuno-oncology. It is expected that the next step in this evolution will involve the integration of ultra-performance liquid chromatography (UPLC) due to its ability to assist in the delivery of branch-specific information on N-glycans involved in the immune response [30].

Future outlook

The groundbreaking work of James Allison and Tasuku Honjo opened new avenues in cancer treatment by unveiling PD-1 and CTLA-4 as therapeutic targets. However, the limitations of these therapies prompted exploration into alternative pathways that tumors use to evade the immune system.

Identifying glycans as both modulators and predictive markers for immunotherapy response is a game changer. Bertozzi's work has marked a pivotal moment in this pursuit, by adapting click chemistry and bioorthogonal chemistry for use in humans. This innovative approach, centered around sialic acid, has broadened the scope of immunotherapy, offering a promising avenue to advance immune checkpoint blockade, bringing glycobiology to the center stage.

Technological advancements, including LC-MS and UPLC allow for precise analysis of sugar profiles, laying the foundation for the development of personalized cancer therapies based on these glycan signatures. By integrating glycobiology into patient stratification approaches, the golden age of molecular-based precision medicine comes ever closer.