Dr. Alejandro Brenes, a Wellcome Trust Early Career Fellow at the University of Edinburgh’s Institute for Regeneration and Repair, is uncovering hidden neutrophil populations in glioblastoma that could reshape how we understand immune responses in brain cancer. His journey from ‘accidental’ researcher to immune cell specialist demonstrates how proteomics can reveal cellular functions invisible to other methods. Through collaborating across disciplines, he’s now focused on developing single-cell proteomics (SCP) as a tool to map out the functions of human immune cells in health and chronic inflammatory disease.

An unexpected journey from Costa Rica to Edinburgh

Alejandro never intended to become a researcher. Growing up in Costa Rica, a country where research funding flows primarily toward biodiversity and marine biology, the idea of pursuing proteomics seemed as distant as the Scottish Highlands he would eventually call home.

“I thought I was coming to Scotland for one year to do a master’s in data science and then leave,” Alejandro recalls. That single year has stretched into a decade-long journey taking him from the University of Dundee to the University of Edinburgh, where he is now a Wellcome Trust Early Career Fellow at the Institute for Regeneration and Repair.

The pivot came about by chance. Fresh from his data science master’s, Alejandro encountered a job opening in Angus Lamond‘s renowned proteomics lab. “Angus was looking for a data scientist to work on proteomics. At that point, I barely remembered anything about proteins, but the opportunity sounded really interesting.”

Unlike many proteomics labs focused on pushing technical boundaries, Lamond’s team was deeply interested in the biology that proteomics could reveal. “Angus had a different focus to Matthias Mann, despite them both starting their labs in offices close to each other at EMBL. Matthias focused on pushing method development to the maximum, Angus focused on how to use proteomics to understand cell biology.”

This focus on the biological knowledge enabled Alejandro to discover his interest for his current research area: the intersection between proteomics and immunology.

A pandemic changes everything

Alejandro had mapped out his PhD trajectory: T cell biology under the guidance of Doreen Cantrell, a pioneering T cell biologist who was behind the first large-scale proteomics study of the adaptive immune system in mice, alongside Lamond’s technical expertise. The plan seemed straightforward until 2020.

“I started my PhD in 2019, shortly after which, the pandemic hit,” Alejandro explains. Suddenly, the newest member of the combined Cantrell-Lamond labs found himself with a target on his back for a complete research pivot. “At that point, they were analysing about 300 patient proteomes of a super exciting cell type. They’re like,

‘Whom can we persuade to shift their research focus onto this new cell type?’”

That researcher turned out to be Alejandro, and the new cell type was the neutrophil – the most abundant immune cell in human blood and one of the body’s first responders to infection and tissue damage. While the redirect seemed daunting, Alejandro quickly discovered that neutrophils were uniquely suited to proteomics analysis, presenting opportunities that other immune cells couldn’t offer.

The neutrophil advantage: when proteomics trumps transcriptomics

Neutrophils develop in the bone marrow, where they’re packed with proteins stored in specialised compartments called granules. By the time they mature and enter circulation, they’ve almost stopped making new proteins. “If you look at mature neutrophils, they have very little mRNA,” Alejandro explains.

This creates a fascinating disconnect between what RNA sequencing reveals and what the cells actually contain. Take neutrophil elastase, a crucial enzyme stored in neutrophil granules. “We estimate that in a mature neutrophil, we can get 40 to 100 million copies of neutrophil elastase protein per cell. And if you look at the transcriptome, you wouldn’t see it.”

This proteome-transcriptome discordance gives neutrophils a unique advantage in the ongoing “why not just do transcriptomics?” debate that plagues many proteomics researchers due to transcriptomics’ maturity as a tool, high throughput, and lower cost.

The technical challenge: small cells, big problems

Despite their biological advantages, neutrophils are understudied as they present formidable technical challenges. They’re tiny, sitting at just 35-60 pg per cell compared to the 250 pg of HeLa cells commonly used in single-cell studies. They’re also “loaded full of proteins called proteases,” which can wreak havoc during sample preparation.

“If you handle them wrongly during sample preparation, you don’t get any tryptic peptides. The proteases easily become active and digest the proteins in loads of unspecific ways before trypsin can do its work” Alejandro notes.

The size limitation was particularly acute when Alejandro began focusing on tissue-specific neutrophil adaptations. Working with clinical collaborators who provide patient samples, means working with whatever you can get. Starting with perhaps 20,000 neutrophils from a biopsy, the purification process might leave a maximum of 10,000 – a number that would have yielded only partial proteome coverage just a few years ago.

The breakthrough came through collaboration with Karl Mechtler’s team in Vienna, specialists in pushing the technical boundaries of SCP. Their combined expertise, along with newer generation instruments and more robust columns transformed what was possible with neutrophil analysis.

“Trying to do neutrophil single-cell proteomics on the Exploris 480 was challenging. We were getting about 180 proteins per cell, at which point there’s little biology that can be discerned,” Alejandro recalls. The switch to the Astral, paired with IonOpticks columns, delivered a dramatic improvement. “We went from 180 to 1,100. With that massive boost, you start to see a lot more about the functions of the cell, about the biology, about their roles and specialisation, even at the single-cell level.”

“With neutrophils, there’s very little leniency. There’s no buffer when things don’t work well. We needed robust columns that could give us the best we could out of the data. It was clear that IonOpticks columns were the ones we need for these complicated samples.”

The enhanced sensitivity enabled Alejandro to pursue increasingly ambitious questions about neutrophil biology across different tissue environments, opening the door to his most surprising findings.

Discovering hidden neutrophil states in brain cancer

But the story didn’t end there. They thought that, as neutrophils spent time in the tumour environment, they began releasing their granules, with the potential to kill tumour cells. Then came an unexpected bifurcation: some neutrophils no longer had the metabolic energy to help you fight the cancer, while others began forming neutrophil extracellular traps (NETs): web-like structures containing DNA, histones, and granules.

“It’s a beautiful acronym NETs,” Alejandro notes. “Neutrophils partially explode and release a sticky web-like structure… with microscopy, you can see this clearly – it’s fascinating. It’s mostly for pathogen control, as it traps bacteria in the sticky web and has granules that kill them.”

Read the full paper here, as well as Alejandro’s reflections on the experience of working on the paper here.

When discovery meets validation

The most intriguing finding was a population of neutrophils that appeared to be stuck in blood vessels, forming NETs uncontrollably. “We think they’re blocking the blood vessels and promoting tumour necrosis,” Alejandro explains. The implications were troubling; this population appeared to help the tumour rather than fight it.

“When we looked at our data for the first time, we wondered, is it correct?” The validation came from an unexpected source: a 2025 Nature publication showing exactly this phenomenon in mouse lung cancer models using 3D imaging.

The convergence was remarkable. “If we look at our proteomic profile and what they describe in the 3D imaging, ours fits beautifully. We met the researcher, Jose, at a conference and saw his work presented. We’re like, ‘wow, this is exciting, that two separate people in different parts of the world, looking at different models, we’re seeing the same thing.’”

Beyond validating each other’s findings, convergent studies like these can be a source of inspiration. “The cool thing with the mouse study is, they found that if you treat these NETs, you seem to reduce tumour necrosis, which could lead to better outcomes,” Alejandro explains. “Maybe this is something we can also explore within the glioblastoma side of things.”

The human side of clinical research

Once the call comes in, the team springs into action. Located just a few hundred meters from the hospital, they can quickly collect samples and begin the intensive processing pipeline: “What we get is a small brain biopsy. And then we do mechanical dissociation of the tissue, digestion and filtering to extract the single cells in solution. Our next step is flow cytometry and eventually mass spec analysis.” The entire process, from surgery to sorted cells ready for proteomics, can take anywhere from five to nine hours.

Alejandro acknowledges that the patients themselves are essential partners in advancing research. “If they didn’t consent, we wouldn’t have been able to do any of this. From patients to the surgeons, the research nurses, and research collaborators, they all form a really important part of the team.”

He also highlights the close collaboration with Karl Mechtler, whose crucial technical expertise was vital for the project. He notes that Karl and, in Karl’s team, Rupert are “phenomenal at pushing the boundaries of what’s possible in mass spectrometry-based SCP.”

“The analysis and interpretation of the data is another huge task.” Alejandro notes. “It can take even longer than the acquisition of the data.” Here, the data science expertise combined with the deep biological knowledge of neutrophils within the senior team in the Walmsley lab enabled them to put the technical results into a biological context.

The expanding horizon and future directions

Alejandro sees SCP approaching a pivotal moment. The field has evolved beyond the exclusive domain of a handful of pioneering laboratories to become technically feasible for a broader range of researchers. “It’s not easy, but it can be done, and it can be done on challenging samples and cells.”

To Alejandro, the real excitement lies in the biological insights that are becoming possible when using SCP. For immunology in particular, SCP offers unique advantages over transcriptomics in understanding cellular function. “With neutrophils, we can see a population that have eaten up cell debris or other cells, we can see degranulation, we can see NETing, and you don’t see those functional states with the transcriptome,” Alejandro explains. He expects similar insights will be possible with T cells, NK cells and macrophages, with SCP significantly expanding our understanding of the immune system.

His own trajectory reflects this expanding horizon. Alejandro has recently been awarded a Wellcome Trust Early Career Award, a prestigious fellowship that will enable him to pursue single-cell proteomic analysis of neutrophils in chronic obstructive pulmonary disease (COPD), the third leading global cause of death in adults over 50. Using the approaches refined in his glioblastoma work, he’ll characterise neutrophil functional states and identify pathogenic populations that drive disease progression.

From rare neutrophil populations in brain tumours to metabolic adaptations in chronic lung disease, single-cell proteomics is revealing the hidden complexity of our immune system one cell at a time, in the tissues where it matters most.

Learn more about Alejandro and his work on his profile