The team, led by senior author Daniel G. Isom, Ph.D., a Sylvester researcher and faculty member in the Department of Molecular and Cellular Pharmacology, turned to a vast computational dataset containing more than 214 million predicted protein structures. Rather than scanning for familiar sequence signatures, the researchers asked a different question: which of these predicted proteins fold into shapes that resemble known functional families, even if their sequences look like nothing recognizable? Their search concentrated on the G protein-coupled receptor family, or GPCRs, an enormous group of membrane proteins that allow cells to sense and respond to signals arriving from outside the cell. GPCRs are among the most heavily exploited targets in pharmacology, so any undiscovered relatives are of more than academic interest.

“For decades, we have largely explored protein biology using sequence as our guide,” Isom said. “We wanted to know what biology we might be missing if we searched by three-dimensional structure instead. What we found suggests there is another layer of biology that has been hiding in plain sight.” The structural approach flagged a set of proteins that classical sequence-based methods had never connected to the GPCR family, a category sometimes described as part of the dark proteome, the substantial fraction of predicted proteins whose functions remain entirely unknown.

One protein in particular stood out. Known as TM184C, it folded like a GPCR, but when the team examined its behavior in cells, it refused to follow the expected script. Classic GPCRs typically sit in the plasma membrane at the cell’s surface, waiting for extracellular ligands. TM184C, by contrast, was found largely inside the cell, embedded in the membranes of intracellular vesicles, the tiny membrane-bound packages that cells use to ferry materials between compartments and to one another. That difference in localization alone hinted that TM184C might represent an entirely new mode of GPCR-like function.

Following the protein’s location in living cells revealed something stranger still. The TM184C-positive vesicles did not sit still. They traveled along microtubules, the rigid protein filaments that serve as the cell’s internal highway system, and they accumulated in thin projections that extend from one cell toward its neighbors. These projections acted like bridges. Through them, the researchers observed cells exchanging metabolites, vesicles, and even entire organelles, including mitochondria, the power-generating structures that supply the energy currency of life. The discovery suggests that direct, physically connected exchange between neighboring cells may be far more common, and far more consequential, than previously appreciated.

“When we saw TM184C-positive vesicles moving through connections between cells, we realized these structures could be routes for substantial material exchange,” said Jenniffer Arcuri, Ph.D., a senior scientist with Sylvester and the study’s lead author. “That completely changed how we thought about TM184C and made us consider how cells might use these connections to cooperate and compete for resources.” To test whether the protein actually mattered, the team disrupted TM184C in cultured cells. The effect was clear: the cells formed fewer intercellular connections, and their overall shape and vesicle organization changed, indicating that TM184C helps build and manage these intercellular conduits rather than merely riding along inside them.

The findings raise a question that cuts to the heart of tissue biology: when neighboring cells share resources, who benefits? In healthy tissue, the exchange could be a form of cooperation, allowing cells under stress to survive by shuttling fuel, building blocks or damaged components to wherever they are needed most. But the conduits could also be exploited. If the exchange is unequal, one cell might gain at another’s expense, drawing support from a weaker neighbor. That possibility becomes especially provocative in cancer, where tumor cells frequently endure low oxygen and scarce nutrients. Intercellular bridges could give some cancer cells a lifeline, allowing them to share resources or siphon support from surrounding tissue in ways that conventional metabolic studies, which typically analyze cells in isolation, would never detect.

“I think cells coordinate until they have to compete,” said Shraddha Chandthakuri, a Cancer Biology doctoral student in the Isom lab. “When the cells are stressed, they may coordinate to redistribute the proteins, organelles, and metabolites to support the survival of the population as a whole.” Bruno Colon, a Molecular and Cellular Pharmacology graduate student in the same lab, is now probing that dynamic directly. “What excites me most is understanding what this exchange actually does to the cells on both sides,” Colon said. “As part of my doctoral work in the Isom lab, I am studying how these connections occur in normal cells and aggressive cancers like glioblastoma. Understanding their role could give us new insight into how these tumors communicate and potentially reveal vulnerabilities we haven’t recognized before.”

TM184C appears to influence more than just physical connectivity. The protein also seems to help regulate autophagy, the recycling program by which cells break down and reuse old or damaged components, a process critical to surviving starvation and other stresses. In the study, when the researchers reduced the amount of TM184C in cells, markers of autophagy rose, suggesting the protein normally acts as a brake or tuning mechanism on the process. Adding a structural dimension to the evidence, the team studied a yeast protein called Hfl1 that resembles the human protein. When Hfl1 was removed from yeast, the cells developed noticeable defects. Remarkably, inserting human TM184C into those yeast rescued the problems, demonstrating that the protein’s essential function has been conserved across roughly a billion years of evolution separating baker’s yeast from humans.

For Isom, the broader lesson is about method as much as mechanism. He emphasized that the work depended on pairing AI-driven structure prediction with rigorous experimental validation, not on trusting the algorithms alone. “AI cannot be blindly trusted, but can lead to really big things in the hands of experts and prepared minds,” he said. “For decades, biomedical research has understandably concentrated on the proteins we could identify and understand. But there is another layer of biology that has remained largely invisible to us. AI gives us a way to start exploring it systematically. TM184C is one example of what can be found when we look.” The implication is that artificial intelligence is not merely accelerating the pace of existing science; it is changing what scientists can see at all. TM184C, pulled out of the dark proteome by searching the shapes rather than the sequences of 214 million predicted proteins, offers both a new way to study how cells communicate, survive stress and possibly drive disease, and a template for finding whatever else has been hiding in plain sight.

Subject of Research: Discovery and functional characterization of the hidden GPCR-like human protein TM184C, which regulates intercellular exchange and autophagy

Article Title: AI helps find hidden human proteins and reveals what they do

Article References: AI helps find hidden human proteins and reveals what they do. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: TM184C, GPCR, dark proteome, artificial intelligence, protein structure prediction, intercellular exchange, autophagy, vesicles, mitochondria, cell-to-cell bridges, Sylvester Comprehensive Cancer Center, cancer biology

The appeal of mEVs begins with their membrane. Cholesterol, sphingomyelin and ceramides help create a relatively robust lipid bilayer that shields internal cargo from environmental damage. Studies summarized in the review indicate that milk vesicles can remain intact under simulated gastrointestinal conditions and protect RNA from digestive enzymes such as ribonucleases. After oral administration, vesicles may interact with intestinal epithelial cells through endocytosis, membrane fusion or receptor-mediated uptake, allowing their contents to enter recipient cells. Some experimental work also suggests that milk vesicles or their cargo can reach tissues beyond the gut, including the brain. This possibility is particularly important because the blood-brain barrier restricts many therapeutic molecules. However, crossing that barrier in animal or cellular models does not establish effective delivery in people. Biodistribution depends on vesicle size, surface proteins, cargo, dose, species of origin, processing conditions and administration route. The review therefore treats gastrointestinal stability and barrier transport as promising properties, not guarantees of therapeutic performance.

Milk vesicles are not a single uniform material. The broader extracellular-vesicle population includes exosomes, microvesicles and apoptotic bodies, which differ in size and how they form. Exosomes develop inside multivesicular bodies when endosomal membranes bud inward to create intraluminal vesicles; multivesicular bodies can then fuse with the plasma membrane and release them. Microvesicles form by outward budding of the cell surface, involving calcium-dependent cytoskeletal changes and redistribution of membrane lipids. In milk, the vesicle population is shaped by the animal species, lactation stage, maternal physiology, diet and health status. Bovine, human, goat, camel, porcine and equine milk can therefore contain different mixtures of proteins and regulatory RNAs. Commonly measured surface or intracellular markers include CD9, CD63, CD81, TSG101 and Alix, but marker detection alone does not define purity or biological function. The review calls for multi-method characterization that combines particle sizing, microscopy, protein analysis, RNA profiling and functional testing.

Obtaining clean vesicles from milk is technically difficult because milk is a complex mixture containing casein micelles, soluble proteins, fat globules and other particles with overlapping physical properties. Differential ultracentrifugation remains widely used, separating material according to size and density, but it can be slow and may promote aggregation or structural damage. Ultrafiltration and polyethylene-glycol precipitation are easier to scale, yet they can recover non-vesicular contaminants. Size-exclusion chromatography separates particles by hydrodynamic size and is often combined with other methods to improve purity. Immunoaffinity capture can select vesicles carrying particular surface markers, although it may reduce recovery and exclude biologically relevant subpopulations. Emerging microfluidic and immunomagnetic systems could enable automated processing of small volumes, while tangential-flow filtration combined with chromatography offers a possible route toward larger-scale production. Across all approaches, researchers need consistent measurements of particle number, size distribution, morphology, membrane integrity, cargo and contaminating milk proteins.

As delivery systems, mEVs could carry molecules that otherwise degrade quickly or dissolve poorly. The review describes experimental loading with polyphenols such as curcumin, resveratrol, quercetin and epigallocatechin gallate, as well as chemotherapeutic compounds including paclitaxel and doxorubicin. Encapsulation may improve aqueous dispersal, protect cargo during digestion and increase contact with intestinal tissues. Vesicles have also been investigated for transporting small interfering RNA, microRNA, messenger RNA, peptides and proteins. In principle, this creates a dual-purpose platform: the vesicle’s native cargo may influence recipient cells, while an added therapeutic molecule supplies a designed activity. Surface engineering could further attach targeting ligands or alter tissue distribution. Yet loading is not straightforward. Passive incubation, membrane permeabilization and other approaches can produce different encapsulation efficiencies and may damage the vesicle. A useful product would require reproducible cargo content, predictable release kinetics and evidence that the loaded molecule reaches the intended tissue at a clinically meaningful dose.

The biological effects reported across models are broad but uneven. In intestinal systems, milk vesicles have been associated with stronger tight junctions, including proteins such as ZO-1, occludin and claudin-1, and with reduced inflammatory signaling. MicroRNAs including miR-148a, miR-21, miR-30a and miR-146b may influence pathways involving NF-κB, Toll-like receptors, DNA methylation and the NLRP3 inflammasome. In cell and animal studies, these mechanisms have been linked to lower inflammatory cytokines, improved barrier function and protection from oxidative stress. Other experiments report effects on macrophage polarization, with some mEV preparations encouraging an anti-inflammatory state. Researchers have also examined bone, liver, heart, lung and pancreatic applications. Milk vesicles have been tested in models of colitis, metabolic dysfunction, fibrosis, osteoporosis, vascular injury and pulmonary inflammation. These findings suggest multiple possible mechanisms, including direct cargo transfer, modulation of gut microbiota and communication along the gut-liver or gut-heart axes, but they do not demonstrate that drinking milk or consuming an unstandardized vesicle preparation treats disease.

Several findings illustrate why careful interpretation is essential. In mice, orally administered milk vesicles have been reported to cross the blood-brain barrier, increase hippocampal dendritic complexity and improve selected cognitive or motor outcomes. Other studies have found changes in microglial DNA-methylation machinery or neuronal survival in cellular models. At the same time, neurological results vary with dose, species, metabolic context and experimental design. A separate line of research has raised hypotheses about interactions between milk exosomes and excessive galactose exposure, while other work found that aging had stronger effects on rat brain lipid profiles and cognition than an extracellular-vesicle-rich supplement. Lung studies likewise include contrasting observations: some mEV preparations protect epithelial barriers or deliver anti-fibrotic compounds, whereas bovine vesicles increased inflammatory macrophage polarization in mice exposed to agricultural dust. In cancer research, vesicles have delivered drugs and gene regulators to tumour models, but one study reported that oral bovine milk vesicles slowed primary-tumour growth while accelerating metastasis. Such results make clear that mEVs are biologically active, context-dependent materials rather than universally beneficial particles.

Translation will depend on proving safety and manufacturing consistency as much as on demonstrating biological activity. Milk origin does not automatically eliminate risk. Preparations can retain caseins, beta-lactoglobulin and other proteins that may trigger reactions in people with milk allergy. Repeated exposure also requires assessment of immune activation, liver and kidney function, oxidative stress, tissue distribution and delayed toxicity. Thermal processing can reduce vesicle yield or disrupt structure, complicating the relationship between fresh milk, pasteurized products and purified formulations. Regulators will also need to determine whether a given product is a food, supplement, biologic, nanomedicine or combination product, since each category carries different requirements. The review points toward standardized isolation protocols, validated vesicle markers, sensitive contaminant testing, single-vesicle and multi-omic analysis, stable storage methods and Good Manufacturing Practice production. Human pharmacokinetic and clinical studies will be decisive. For now, milk-derived extracellular vesicles represent a promising natural nanocarrier platform whose future rests on converting intriguing laboratory observations into reproducible, well-controlled and demonstrably safe interventions.

A further advantage of mEVs is that their value may extend beyond their role as passive containers. Their membranes carry naturally occurring adhesion molecules, tetraspanins and transport-related components that can influence how vesicles are recognized, internalized and distributed. This biological interface distinguishes them from liposomes and polymeric nanoparticles, whose composition can be tuned with considerable precision but generally requires deliberate surface engineering to achieve comparable interactions with cells. The contrast is not absolute: synthetic systems offer stronger control over particle uniformity, drug loading and release kinetics, while mEVs offer a more physiologically integrated membrane and a potentially favorable safety profile. Hybrid designs that combine EV membranes with synthetic cores therefore represent one strategy for balancing biological compatibility with manufacturing control.

The native cargo also complicates how mEV products should be designed and evaluated. A preparation intended to deliver an added drug or RNA may simultaneously contain endogenous proteins, lipids, microRNAs and metabolites capable of altering immune or metabolic responses. Those constituents could contribute to efficacy, but they could also vary with animal species, lactation conditions, feed, health status and processing history. Consequently, measuring total particle concentration is insufficient for comparing products. Functional potency assays will need to establish whether a defined preparation produces a reproducible cellular response, while molecular profiling can help identify which cargo components are retained, enriched or lost during isolation and loading. This is especially important when the desired activity depends on cooperation between the vesicle membrane and its internal contents rather than on a single therapeutic molecule.

Manufacturing scale is promising but should not be confused with readiness for routine clinical use. Milk provides a comparatively accessible starting material, and the review describes ultracentrifugation, size-exclusion chromatography and precipitation methods as established approaches, with tangential-flow and related technologies offering routes toward process intensification. At larger scale, however, purification must preserve membrane integrity while removing abundant non-vesicular milk constituents and maintaining consistent particle and cargo characteristics. The field has reached early translational milestones, including a reported first clinical trial involving mEV-based formulations for RNA therapeutics and anticancer agents, but such studies are only an initial test of feasibility. Results from carefully controlled human investigations will need to define dose, absorption, biodistribution, immune effects and clinically meaningful benefit before the farm-to-pharmacy concept can support approved interventions.

Subject of Research: Milk-derived extracellular vesicles as nutritional and therapeutic nanocarriers

Article Title: Milk-derived extracellular vesicles: nutritional significance, nano-delivery potential, and emerging therapeutic applications – an updated review

Article References: Wang, S., Shaukat, A., Al-Rasheed, M., Tareen, A. M., Arain, M. A., & Luo, C. (2026). Milk-derived extracellular vesicles: nutritional significance, nano-delivery potential, and emerging therapeutic applications – an updated review. Food Science of Animal Resources, 46(1), Article 96. https://doi.org/10.1007/s44463-026-00104-6

Image Credits: AI Generated

DOI: 10.1007/s44463-026-00104-6

Keywords: extracellular vesicles, milk nanocarriers, nutraceutical delivery, drug delivery, microRNAs, precision medicine, gut health, nanomedicine, Milk-derived, extracellular, vesicles, nutritional