Tumor-associated macrophages and microglia collectively constitute one of the largest immune cell populations in glioblastoma, often accounting for up to a third of the total cellular mass of the tumor. Unlike the inflammatory macrophages that would normally sweep into tissue to destroy pathogens or clear damaged cells, macrophages residing inside glioblastomas adopt a profoundly tumor-supportive identity. They secrete growth factors that stimulate tumor cell proliferation, remodel the extracellular matrix in ways that ease invasion, suppress cytotoxic T cell activity, and blunt the effectiveness of immunotherapies that have transformed the treatment of many other cancers. Understanding where these macrophages come from, and what forces shape their immunosuppressive character, has therefore become a central question in neuro-oncology.

The prevailing view has long been that tumor-associated macrophages in the brain arise from two principal sources. The first is the resident microglia, the innate immune cells native to the central nervous system, which become corrupted by tumor-derived signals. The second is circulating monocytes, produced in the bone marrow, which are recruited across the disrupted blood-brain barrier and then differentiate into macrophages within the tumor microenvironment. The new study focuses on this second pathway and asks a deceptively simple question: what cellular intermediaries in the tumor decide that an incoming monocyte should become a macrophage, and what kind of macrophage it becomes?

The answer, the researchers report, involves a population of pericyte-like stromal cells that express fibroblast activation protein, a membrane-bound serine protease that has served for decades as a marker of activated fibroblasts in wound healing and in the desmoplastic stroma of many solid tumors. Pericytes are mural cells that normally wrap around blood vessel endothelial cells, stabilizing vasculature and helping to maintain the blood-brain barrier. In glioblastoma, however, the study indicates that a subset of these pericyte-like cells acquires FAP expression and, with it, a striking new function: the ability to promote the differentiation of monocytes into macrophages with a tumor-associated phenotype.

Technically, the investigators combined single-cell transcriptomic analysis with functional assays to dissect this interaction. Single-cell RNA sequencing allows researchers to profile the gene expression of thousands of individual cells within a tumor, revealing not only the identity of rare cell populations but also the signaling ligands and receptors they deploy. By mapping the communication networks among tumor cells, macrophages, and stromal compartments, the team was able to identify FAP-positive pericyte-like cells as a hub of immunomodulatory signaling. In co-culture experiments, these cells were shown to drive monocytes toward a macrophage fate, biasing the resulting cells toward the immunosuppressive, pro-tumoral polarization that characterizes tumor-associated macrophages in glioblastoma.

The clinical implications of this finding are considerable. Glioblastoma has proven stubbornly resistant to immune checkpoint inhibitors, the antibody therapies that unleash T cells against melanoma, lung cancer, and many other malignancies. One widely cited explanation is that the glioblastoma microenvironment is saturated with immunosuppressive macrophages and microglia that actively paralyze T cells. If FAP-positive pericyte-like cells are among the architects of this immunosuppressive army, then targeting them, or the signals they use to educate monocytes, could represent a way to thin the ranks of tumor-associated macrophages and thereby open a window for T cell-based immunotherapy to function.

Fibroblast activation protein itself is an appealing therapeutic target. It is minimally expressed in healthy adult tissues but abundant in activated stromal cells across multiple cancers, which has already made it the focus of antibody-drug conjugates, small-molecule inhibitors, and radioligand imaging agents in ongoing clinical trials elsewhere in oncology. The demonstration that FAP marks a functionally important stromal population within glioblastoma raises the possibility that strategies developed for pancreatic, breast, and colorectal cancers could be adapted to brain tumors, although the blood-brain barrier and the infiltrative nature of glioblastoma present formidable delivery challenges that any such approach would need to overcome.

Beyond therapy, the study adds an important conceptual layer to the evolving understanding of the glioblastoma microenvironment. Pericytes have traditionally been studied for their roles in vascular biology: they regulate capillary diameter, contribute to blood-brain barrier integrity, and scavenge cellular debris. The idea that a pericyte-derived population can act as an immune educator, converting myeloid precursors into tumor-promoting macrophages, underscores how fluid the functional boundaries are between the vascular, stromal, and immune compartments of a tumor. It also helps explain why glioblastomas are so consistently and comprehensively immunosuppressive: the tumor appears to recruit or reprogram multiple cell types, including structural cells of its own vasculature, into a coordinated anti-immune apparatus.

There remain important questions for future work. The precise molecular signals by which FAP-positive pericyte-like cells induce monocyte differentiation, whether through secreted cytokines such as colony-stimulating factors, direct cell-cell contact, or remodeling of the extracellular matrix, will determine which points in the pathway are most druggable. It will also be essential to establish how these cells are themselves generated, whether they represent a distinct developmental lineage or a pathological reprogramming of ordinary pericytes, and whether their abundance correlates with patient outcomes, treatment resistance, or response to emerging immunotherapies. Longitudinal studies in patient cohorts and validation in additional model systems will be needed to translate the mechanism into prognostic and therapeutic tools.

Nevertheless, the study offers a vivid illustration of how modern single-cell biology is dismantling the old picture of tumors as homogeneous masses of malignant cells. Glioblastoma emerges instead as an ecosystem, one in which tumor cells, immune cells, vascular cells, and stromal cells engage in continuous negotiation, with each population reshaping the others for the tumor’s benefit. Identifying FAP-positive pericyte-like cells as promoters of macrophage differentiation adds a new node to this ecosystem map, and with it, a new set of potential targets. For patients facing a disease with a median survival measured in months despite surgery, radiation, and chemotherapy, every new node represents a new opportunity, and this one connects two of the most immunologically important cell types in the tumor. The hope, shared across the field, is that disrupting this stromal-immune axis could finally give immunotherapy a foothold in one of medicine’s most stubborn cancers.

The distinction between microglia and monocyte-derived macrophages has become increasingly tractable in recent years thanks to advances in single-cell and fate-mapping techniques. Microglia carry a transcriptional signature shaped by their embryonic origin and lifelong residence in the central nervous system, while recruited macrophages retain markers of their bone marrow lineage. Being able to separate these populations reliably matters therapeutically, because the two compartments respond differently to environmental cues and may require different intervention strategies. The identification of a stromal intermediary that actively shapes the monocyte-derived arm adds a layer of specificity to this growing taxonomy.

Pericyte plasticity is itself an area of intense investigation. In models of tissue injury, pericytes can detach from vessels, adopt migratory and secretory behaviors, and participate in scar formation. Similar programs appear to be activated within tumors, where aberrant signaling from malignant cells and from the disrupted vasculature may push pericytes into states that diverge substantially from their physiological roles. The acquisition of fibroblast activation protein expression by pericyte-like cells in glioblastoma fits this broader pattern of stromal remodeling, suggesting that the tumor co-opts a wound-healing-like program for its own purposes.

It is also worth noting that the monocyte-to-macrophage transition is not a single step but a continuum, with intermediate cells that retain plasticity. Signals encountered during recruitment and early differentiation can lock in long-lasting epigenetic programs, meaning that brief exposure to stromal factors may have durable consequences for macrophage behavior. This temporal sensitivity creates potential intervention windows: if the educational signal from FAP-positive cells can be interrupted early, the resulting macrophages might never adopt their tumor-supportive identity.

Finally, the convergence of stromal biology and immunology reflected in this work mirrors a trend across oncology, where cancer-associated fibroblasts and other stromal elements are increasingly recognized as active participants in immune evasion rather than passive scaffolding, reinforcing the case for combination approaches that target both malignant and stromal compartments.

Subject of Research: FAP-positive pericyte-like cells promoting monocyte differentiation into tumor-associated macrophages in glioblastoma

Article Title: FAP+ pericyte-like cells promote monocyte differentiation into tumor-associated macrophages in glioblastoma

Article References: Houdova Megova, M., Shard, C., Vymolova, B., Ternerova, N., Svablova, T., Buna, T., Straka, D., Vanickova, Z., Krepela, E., Kupcova Skalnikova, H., Kolar, M., Sachova, J., Kubovciak, J., Balaziova, E., Vymola, P., Hrabal, P., Ebert, L. M., Patil, A., Tomas, R., … Sedo, A. (2026). FAP+ pericyte-like cells promote monocyte differentiation into tumor-associated macrophages in glioblastoma. Experimental & Molecular Medicine. https://doi.org/10.1038/s12276-026-01827-8

Image Credits: AI Generated

DOI: 10.1038/s12276-026-01827-8

Keywords: glioblastoma, tumor-associated macrophages, pericytes, FAP, monocytes, tumor microenvironment, immunosuppression, single-cell sequencing, immunotherapy, neuro-oncology, pericyte-like, cells

Immune cells are particularly instructive subjects for this kind of analysis because their function depends on rapid, spatially organized surface events. A T cell encountering an antigen-presenting cell reorganizes its membrane within minutes, clustering receptors and adaptor proteins into a structured interface known as the immunological synapse. B cells undergo analogous rearrangements when their B cell receptor binds antigen. Natural killer cells survey target cells and form activating or inhibitory contacts whose outcomes depend on the balance of receptor signals at the contact site. In each case, the physical state of the membrane at the interface is not incidental; it determines which proteins can diffuse into or out of the contact zone, which lipid species segregate there, and how efficiently the cytoskeleton can be remodeled to stabilize or dissolve the interaction.

The biophysical basis of membrane order lies in the composition and behavior of the lipid bilayer itself. Sphingolipids and phospholipids with saturated acyl chains pack tightly and adopt extended conformations, producing regions of high order. Unsaturated phospholipids, with kinks introduced by double bonds, disrupt packing and lower local order. Cholesterol intercalates between phospholipids and has a concentration-dependent effect: at moderate levels it rigidifies fluid bilayers and promotes the coalescence of ordered domains, while at high levels it can increase order further in saturated lipid environments. These interactions underlie the long-standing hypothesis of lipid rafts, nanoscale assemblies enriched in sphingolipids, cholesterol, and certain lipid-anchored proteins that have been proposed to serve as platforms for signaling. Direct visualization of rafts in living cells proved technically elusive for decades because the domains are small, transient, and below the diffraction limit of conventional microscopy, which fueled considerable debate about their physiological relevance.

Probe-based imaging has been central to resolving this debate. Environmentally sensitive dyes such as laurdan and its derivatives report on the hydration and packing of their lipid surroundings through shifts in their emission spectra, allowing order to be quantified as a generalized polarization value. When such probes are targeted to specific leaflets of the plasma membrane or conjugated to molecules that partition preferentially into ordered or disordered phases, they provide spatially resolved readouts of membrane physics in live cells. The interpretation of these measurements requires care, because probe partitioning can itself perturb the membrane, and spectral readouts can be confounded by factors such as pH, probe concentration, and photobleaching. Advances in probe chemistry, calibration standards, and imaging modalities have progressively addressed these concerns, making it possible to compare order measurements across cell types and experimental conditions with increasing confidence.

Super-resolution microscopy techniques have further transformed the field by bringing the relevant length scales within reach. Stimulated emission depletion microscopy, photoactivated localization microscopy, and stochastic optical reconstruction microscopy each achieve effective resolutions well below the diffraction limit, revealing that proteins and lipids once thought to be uniformly distributed actually occupy discrete nanoscale clusters. Combining these structural methods with spectral imaging of order-sensitive probes allows researchers to ask whether regions of high membrane order coincide with clusters of signaling proteins, and whether such coincidence changes upon receptor activation. In immune cells, this combination has shown that ordered domains accumulate at sites of receptor engagement and that disrupting ordered lipid phases, for example by depleting cholesterol or inhibiting sphingolipid synthesis, impairs signaling outputs such as calcium flux, phosphorylation cascades, and cytokine production.

The relationship between membrane order and the actin cytoskeleton adds another layer of regulatory complexity. Cortical actin filaments exert forces on the overlying membrane, creating regions of tension and constriction that can influence lipid phase behavior. Actin-driven structures such as membrane ruffles, microvilli, and picket-and-fence arrangements compartmentalize lateral diffusion, effectively corralling proteins and lipids into transient domains. Conversely, the lipid composition of the membrane affects how actin-binding proteins attach to the cytoplasmic face, creating a bidirectional feedback loop. In migrating immune cells, leading-edge membranes enriched in unsaturated lipids and low order support the protrusive activity needed for chemotaxis, while the uropod exhibits different physical properties that promote adhesion and retraction. Mapping order across a polarized cell therefore reveals how physical heterogeneity aligns with functional polarity.

Pathogens have evolved to exploit membrane physical properties during infection, which underscores the selective pressures shaping these systems. Enveloped viruses bud from membranes whose lipid composition facilitates assembly and release, and some viruses preferentially incorporate ordered lipid domains into their envelopes. Bacterial toxins that bind cholesterol or sphingomyelin use ordered domains as points of attachment for pore formation. Intracellular pathogens manipulate host membrane traffic and lipid metabolism to create replication niches with altered physical properties. In each scenario, the immune response must contend with a membrane environment that the pathogen has actively reshaped, and measurements of membrane order in infected cells can reveal these manipulations as measurable shifts in surface biophysics.

Aging and metabolic state also leave imprints on membrane order. Dietary lipid composition influences the saturation profile of membrane phospholipids over time, and age-associated changes in lipid metabolism have been documented in immune cells from multiple organisms. Membranes from aged T cells, for example, show altered cholesterol content and modified order characteristics that correlate with diminished signaling capacity upon antigen stimulation. Metabolic diseases such as obesity and diabetes, which alter circulating lipid profiles, produce measurable changes in the membrane properties of circulating leukocytes. These observations suggest that membrane order could serve as an integrative readout of an organism’s metabolic and inflammatory history, encoded in the physical state of its immune cell surfaces.

Therapeutically, the sensitivity of membrane order to lipid metabolism opens avenues for intervention. Statins, which reduce cholesterol synthesis, have immunomodulatory effects that may partly reflect changes in membrane organization. Drugs targeting sphingolipid metabolism, such as inhibitors of sphingomyelin synthase or glucosylceramide synthase, alter ordered domain abundance and have shown effects on inflammatory signaling. Fingolimod, a sphingosine-1-phosphate receptor modulator used in multiple sclerosis, acts in part through receptor internalization but also engages with the broader biology of sphingolipid-enriched membranes. Understanding how such agents redistribute membrane order across immune cell subsets could explain some of their off-target effects and guide the design of compounds that tune immune responses through membrane biophysics rather than direct receptor antagonism.

Methodological standardization remains an important challenge for the field. Different probes report on different aspects of membrane physics, and values obtained with one dye are not directly comparable to those from another without careful cross-calibration. Sample preparation, temperature, imaging parameters, and analysis pipelines all influence measured values, and the field has not yet converged on universally accepted reference standards. Efforts to establish standardized protocols, share calibration reagents, and report measurements in ways that facilitate comparison across laboratories will be essential if membrane order is to mature from a research measurement into a reproducible biomarker. The application of machine learning approaches to extract order-related features from large imaging datasets may also accelerate progress by identifying patterns that manual analysis would miss.

The diversity of immune cell subsets presents both an opportunity and a complication. Myeloid cells, lymphocytes, and innate lymphoid cells each maintain distinct lipidomes shaped by their developmental programs and functional demands. Within a single subset, activation state, differentiation stage, and tissue microenvironment further modify membrane composition. A dendritic cell maturing in response to pathogen-associated molecular patterns remodels its membrane as part of its transition from antigen capture to antigen presentation. Tissue-resident macrophages adapt their membrane properties to the lipid milieu of their organ of residence, which differs substantially between brain, lung, liver, and adipose tissue. Comprehensive maps of membrane order across this diversity would require systematic sampling, but the resulting atlas could reveal how physical membrane states encode functional specialization in ways that transcriptomic or proteomic measurements alone do not capture.

Looking forward, the integration of membrane order measurements with other single-cell modalities promises a more complete picture of immune regulation. Combining order imaging with live-cell reporters of signaling activity, such as fluorescent biosensors for kinase activity or calcium, would allow direct testing of causal relationships between membrane physics and signal transduction at the single-cell level. Pairing order measurements with lipidomics would connect physical readouts to their molecular determinants. Spatial transcriptomics and proteomics of tissue sections could place membrane biophysical states in their anatomical and pathological contexts. As these datasets accumulate, the plasma membrane’s physical organization may come to be recognized as a fundamental layer of cellular regulation, one that immune cells exploit with particular sophistication and one that offers distinct targets for therapeutic modulation of immunity.

Subject of Research: Plasma membrane order maps functional diversity in immune cells

Article Title: Plasma membrane order maps functional diversity in immune cells

Article References: Andronico, L. A., Gurdap, C. O., Arora, A., Ragaller, F., Sandoz, P. A., Jiang, Y., Giatrellis, S., de Boer, L. L., Carannante, V., Iskrak, S., Mikes, J., Buggert, M., Österborg, A., Önfelt, B., Klymchenko, A. S., Brodin, P., & Sezgin, E. (2026). Plasma membrane order maps functional diversity in immune cells. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02322-x

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02322-x

Keywords: Plasma, membrane, order, maps, functional, diversity, immune, cells, scientific research

PCOS is one of the most common endocrine disorders affecting women of reproductive age, characterized by irregular ovulation, clinical or biochemical signs of elevated androgens, and the presence of polycystic ovarian morphology. A hallmark of the condition is an excess of small, arrested follicles that fail to reach developmental maturity, a phenomenon known as follicular arrest. Anti-Müllerian hormone, or AMH, is often elevated in PCOS patients because of the abundance of small growing follicles, and it has become a valuable biomarker for diagnosis and disease severity. Yet the cellular reasons why these follicles stall remain incompletely understood. Because granulosa cells supply the developing follicle with energy, growth factors, and vascular signals, they represent a logical place to look for the roots of this arrest.

To investigate, the team isolated granulosa cells from women undergoing in vitro fertilization at a single center, applying the Rotterdam criteria to diagnose PCOS. The final cohort consisted of a control group of twelve women whose serum AMH levels fell within the normal range of 2 to 5 nanograms per milliliter, and a PCOS group of eleven women who met the Rotterdam criteria and displayed elevated AMH above 5 nanograms per milliliter. To control for the possibility that differences might simply reflect follicle size rather than disease, the researchers further subdivided cells from both groups according to follicular diameter, comparing cells from large follicles exceeding 14 millimeters with those from small follicles under 14 millimeters. All cells were cultured under standardized laboratory conditions, allowing the team to compare morphology, proliferation, mitochondrial activity, and secretory function directly.

The results were consistent across several independent lines of measurement. Under the microscope, PCOS-derived granulosa cells displayed abnormal morphology and an enlarged cell size compared with cells from healthy controls. When their capacity to divide was assessed, the PCOS cells proliferated significantly more slowly. This impaired growth is particularly consequential because granulosa cell proliferation drives follicle expansion during development; cells that cannot multiply properly cannot support a follicle’s progression toward ovulation. The finding suggests that the follicular arrest characteristic of PCOS may begin within the somatic compartment of the follicle rather than being solely an oocyte problem.

Deeper analysis revealed where the cellular failure likely originates: the mitochondria. These organelles serve as the cell’s power plants, generating adenosine triphosphate, or ATP, the chemical currency that fuels virtually every energy-demanding process, including cell division, protein synthesis, and secretion. The researchers found that both mitochondrial function and intracellular ATP levels were significantly reduced in PCOS granulosa cells. This energy deficit provides a coherent explanation for the observed proliferation defect, as cells with insufficient ATP cannot sustain the biosynthetic workload required to replicate. Mitochondrial dysfunction in granulosa cells has been suspected in PCOS before, but linking it quantitatively to both proliferative failure and secretory impairment in the same cohort strengthens the case that it is a central defect rather than an incidental finding.

Perhaps the most novel component of the study concerns angiogenesis, the formation of new blood vessels, which is essential for follicle development. A growing follicle depends on a rich vascular network to receive oxygen, nutrients, and hormones from the bloodstream. Granulosa cells contribute to building this network indirectly through paracrine signaling, releasing factors that stimulate nearby endothelial cells to organize into vessel structures. To test this function, the team collected conditioned media, essentially the liquid culture environment in which the granulosa cells had been growing, and applied it to human umbilical vein endothelial cells in a tube formation assay, a standard laboratory test of angiogenic capacity. The conditioned media from PCOS granulosa cells significantly impaired the ability of endothelial cells to form tubes, demonstrating that the angiogenic support normally provided by these ovarian cells was diminished in the disease state.

The effect was not uniform across follicle sizes. Granulosa cells harvested from larger follicles showed a more pronounced impairment in angiogenic support than those from smaller follicles, an observation that could help explain why larger follicles in PCOS ovaries so often fail to progress to ovulation despite reaching substantial size. At the molecular level, the researchers examined the expression of angiogenesis-related cytokines and found that three key pro-angiogenic chemokines, CXCL6, IL8, and MCP1, were consistently downregulated in PCOS granulosa cells. Interestingly, vascular endothelial growth factor A, or VEGF-A, the most famous angiogenic factor, showed a less consistent pattern, suggesting that the angiogenic deficit in PCOS is not simply a matter of reduced VEGF but rather a broader disruption of the chemokine-mediated signaling network that coordinates blood vessel formation.

Taken together, the findings sketch a coherent mechanistic framework for how PCOS disrupts follicle development. Mitochondrial dysfunction reduces ATP availability, which in turn limits cellular proliferation and dampens the secretion of angiogenic chemokines. Reduced angiogenic signaling compromises the vascular supply to developing follicles, depriving both the granulosa cells and the oocyte of the metabolic support needed for maturation. The authors describe this as a functional interplay between metabolic dysfunction and disrupted chemokine-mediated angiogenic signaling, a chain of causation that connects the metabolic phenotype of PCOS to its reproductive consequences. Because the chemokines CXCL6, IL8, and MCP1 emerged as consistently downregulated factors, they represent plausible targets for interventions aimed at restoring follicular vascular support in affected women.

The study carries practical implications for fertility medicine. Many women with PCOS require assisted reproductive technology to conceive, and the quality of the follicular environment is a determinant of oocyte competence and embryo development. If the granulosa cell dysfunction identified here proves to be modifiable, strategies to improve mitochondrial function or replenish angiogenic chemokine signaling could theoretically enhance follicle quality in PCOS patients undergoing IVF. Such approaches remain speculative, and the study is a relatively small observational analysis conducted at a single center, so the findings will need replication in larger and more diverse cohorts before they translate into clinical protocols. The authors note that the work provides potential targets for improving reproductive outcomes rather than an immediate treatment.

Beyond its clinical relevance, the study contributes to a growing appreciation of the ovary as a metabolically demanding organ in which cellular energy status and developmental signaling are tightly intertwined. The follicle is often studied primarily through its hormonal and genetic regulation, but this research underscores that the physical infrastructure of follicle growth, from mitochondrial ATP production to the surrounding vasculature, may be equally decisive. For the millions of women living with PCOS worldwide, a condition that remains among the leading causes of anovulatory infertility, understanding that their follicles may be starved of both energy and vascular support offers a new dimension to the search for causes and cures. As research continues to map the molecular pathways linking mitochondrial health, chemokine signaling, and folliculogenesis, the granulosa cell may well emerge as a key gateway through which future therapies for PCOS are delivered.

The study was conducted under ethical oversight at Cathay General Hospital in Taipei, with approval from the hospital’s Ethics Committee and written informed consent obtained from all participants, in accordance with the Declaration of Helsinki. The work received financial support from the National Science and Technology Council of Taiwan and from Cathay General Hospital, and the authors declared no competing interests.

Readers should note that the article was published as an accepted manuscript in open access form, released early to provide faster access to peer-reviewed research. This version is citable and carries a permanent DOI, though it remains subject to editorial revisions before the final Version of Record replaces it. The research is categorized under topics including endocrine reproductive disorders, fertility, and gonadal disorders, reflecting its position at the intersection of reproductive endocrinology and cellular metabolism research.

Subject of Research: Mitochondrial dysfunction in granulosa cells is associated with impaired proliferation and angiogenic support in women with polycystic ovarian syndrome and elevated AMH

Article Title: Mitochondrial dysfunction in granulosa cells is associated with impaired proliferation and angiogenic support in women with polycystic ovarian syndrome and elevated AMH

Article References: Hong, K.-J., Lin, J.-J., & Lai, T.-H. (2026). Mitochondrial dysfunction in granulosa cells is associated with impaired proliferation and angiogenic support in women with polycystic ovarian syndrome and elevated AMH. Journal of Ovarian Research. https://doi.org/10.1186/s13048-026-02264-x

Image Credits: AI Generated

DOI: 10.1186/s13048-026-02264-x

Keywords: Mitochondrial, dysfunction, granulosa, cells, associated, impaired, proliferation, angiogenic, support, women, polycystic, ovarian

BEST4⁺ cells are defined by a distinctive combination of genes, including BEST4, OTOP2, CA7 and GUCY2C. In the small intestine, they also show particularly high expression of CFTR, the chloride and bicarbonate channel best known for its role in cystic fibrosis. Together, these markers suggest a specialized role in moving negatively charged ions across the epithelium. Chloride and bicarbonate transport draw water into or out of the intestinal lumen and help regulate the chemical environment surrounding epithelial cells and mucus. OTOP2 adds a sensing capability: this proton-selective channel can respond to changes in extracellular acidity. CA7 may support intracellular bicarbonate production, while GUCY2C encodes guanylyl cyclase C, a receptor that converts extracellular signals into cyclic GMP. The resulting molecular profile is unusually coherent for a relatively small cell population.

The cells were first associated with the intestinal epithelium through studies of bestrophin expression, but modern single-cell sequencing made their identity much clearer. These analyses separate individual epithelial cells according to their RNA profiles, allowing researchers to identify populations that conventional staining can overlook. BEST4⁺ cells appear early in human intestinal development, reportedly as early as gestational week 11, and generally represent less than 5 percent of the fetal epithelial compartment. In adults, their abundance varies by region, with reported enrichment in parts of the jejunum, ileum and colon. Small-intestinal cells are concentrated toward the upper and middle villus, whereas colonic cells tend to occupy apical crypt regions. The regional differences suggest that a shared core program may be adapted to local pH, microbial exposure, mucus and transport demands.

The developmental identity of BEST4⁺ cells is still being debated. Several lines of evidence place them near the end of an absorptive differentiation pathway. They occupy post-mitotic compartments, lack conventional proliferation markers and express genes associated with mature enterocytes and colonocytes, including VIL1, AQP8 and SLC26A3. Human organoid experiments indicate that NOTCH signaling is required for their emergence, and the transcription factor SPIB appears indispensable: removing SPIB with CRISPR-based gene editing prevented BEST4⁺ cell generation even when NOTCH signaling remained active. Yet other findings point toward connections with the secretory lineage. Trajectory analyses in human tissue have identified a low-probability link to ATOH1-positive secretory progenitors, while zebrafish lineage-tracing experiments suggest that related cells can arise from secretory precursors. These differences may reflect species, anatomical region or inflammatory state rather than a single universal developmental route.

Under normal conditions, BEST4⁺ cells may act as coordinated pH and ion-transport units. BEST4 belongs to the bestrophin family of calcium-activated anion channels, which can conduct chloride and bicarbonate when intracellular calcium rises. CFTR provides another major route for apical anion secretion. The cells also express guanylin and uroguanylin, the endogenous ligands for GUCY2C, alongside the receptor itself. This arrangement could create an autocrine circuit in which locally produced ligands stimulate cyclic GMP, activate downstream protein kinases and increase CFTR activity. Bicarbonate secretion is important beyond fluid balance: it helps neutralize acidity near the epithelial surface and allows newly released MUC2 mucin to hydrate and expand into an effective protective layer. The review therefore proposes that BEST4⁺ cells may support mucus-barrier assembly in cooperation with goblet cells, although direct proof that these cells provide the critical bicarbonate flux is still lacking.

The same machinery can be exploited during secretory diarrhea. Bacterial heat-stable enterotoxin, or STa, binds and activates GUCY2C, raising intracellular cyclic GMP and stimulating CFTR through protein kinase G. Cholera toxin and the heat-labile toxin of enterotoxigenic Escherichia coli activate adenylate cyclase through persistent cyclic AMP signaling, leading to protein kinase A-mediated CFTR activation. In both cases, excessive chloride and bicarbonate secretion promotes water movement into the lumen. The review highlights evidence that BEST4⁺ cells are unusually equipped for this response because they co-express GUCY2C and CFTR at functionally relevant levels. Human intestinal organoids exposed to these pathways swell as fluid accumulates. Investigational inhibitors of GUCY2C or CFTR can reduce secretion in experimental systems, but broad suppression carries risks because basal GUCY2C signaling also contributes to barrier integrity, mucus hydration and epithelial maintenance.

Viral diarrhea may involve BEST4⁺ cells more indirectly. Porcine epidemic diarrhea virus preferentially damages villus absorptive enterocytes and can impair NHE3, a sodium-hydrogen exchanger needed for sodium-coupled water absorption. Studies in infected piglets have reported reduced expression of several water and nutrient transporters, together with increased ileal CFTR transcripts. Electrical measurements of infected jejunal tissue also indicate enhanced secretory responses. These observations are consistent with a shift away from absorption and toward secretion, and the presence of CFTR-rich BEST4⁺ cells makes them plausible contributors. But the available evidence does not show that the virus infects BEST4⁺ cells or that the cells are responsible for the altered current. Rotavirus offers another possible route: its NSP4 protein disturbs calcium signaling and triggers ADP-dependent calcium waves in neighboring uninfected cells. BEST4⁺ cells could respond as bystander effectors through calcium-sensitive anion channels, but this remains a testable hypothesis.

Norovirus likewise causes changes that could intersect with the BEST4⁺ program, including reduced epithelial resistance and increased electrogenic chloride secretion. The virus can replicate in differentiated enterocytes and some enteroendocrine cells, but there is no direct evidence that mature BEST4⁺ cells support norovirus replication. Inflammation may nevertheless alter their numbers or activity. In human organoids, interferon-gamma increases BEST4⁺ cell differentiation through a SPIB-dependent mechanism, and the resulting cells show stronger CFTR-dependent secretion after toxin exposure. If antiviral inflammation produces a similar response in living intestine, it could amplify fluid loss during acute disease. Conversely, bicarbonate secretion and mucus hydration might aid barrier repair during recovery. The review also connects BEST4⁺ cells to inflammatory bowel disease, where their abundance and expression of transport and metal-buffering genes appear altered, and to cystic fibrosis, in which defective CFTR trafficking may leave these high-CFTR cells unable to regulate anion transport, luminal acidity and mucus hydration.

These possibilities make BEST4⁺ cells attractive but challenging therapeutic targets. A drug that blocks pathological GUCY2C or CFTR activation could reduce fluid loss, yet permanent or systemic inhibition might undermine normal mucosal defense. Experimental CFTR inhibitors have reduced toxin-induced secretion in rodents, although some show limited solubility, rapid washout, narrow dosing windows or off-target effects on mitochondria. A more selective strategy may involve ADRA2A, an adrenergic receptor enriched in human BEST4⁺ cells; activating it suppresses cyclic AMP secretion and reverses cholera-toxin-induced swelling in enriched organoids. Such findings remain preclinical. The next steps will require lineage-specific genetic tools, direct electrophysiological measurements and disease experiments in animals that actually possess a conserved BEST4⁺ population. Conventional laboratory mice lack a canonical intestinal Best4 lineage, making rats, pigs, zebrafish and human organoids complementary rather than interchangeable models. Until researchers can manipulate these cells in vivo, BEST4⁺ cells should be viewed not as a confirmed master switch for viral diarrhea, but as a promising framework for understanding how infection, inflammation and epithelial ion transport converge.

At the molecular level, the proposed hub function depends on the way several transport systems are colocated rather than on BEST4 alone. Bestrophin channels are described as pentameric calcium-sensitive anion channels with a calcium-binding region, a hydrophobic gate and a cytoplasmic regulatory segment. This architecture provides a potential link between intracellular calcium signals and rapid changes in chloride or bicarbonate permeability. In a BEST4⁺ cell, such calcium-dependent conductance could complement CFTR, whose activity is controlled primarily through cyclic-nucleotide signaling. The two routes therefore offer distinct but potentially convergent means of regulating apical anion movement, while OTOP2 and CA7 could help couple that transport activity to the chemical conditions at the epithelial surface.

That arrangement also helps explain why anatomical location matters. BEST4⁺ cells are reported in the proximal small intestine and at the colonic surface, but their associated transport programs are not identical in every region or species. Villus-associated small-intestinal cells encounter different nutrient, acid and fluid gradients from cells near colonic crypt openings. Cross-species conservation supports a shared cellular program, yet conservation of marker genes does not establish conservation of net ion flux. Differences in epithelial architecture, microbiota, mucus organization and channel abundance could alter the physiological contribution of the same transcriptional cell type. Functional comparisons will therefore need to measure transport in defined regions rather than treating all BEST4⁺ cells as equivalent.

A central experimental challenge is separating correlation from cell-specific causation. High BEST4, GUCY2C or CFTR expression identifies a candidate effector population, but whole-organoid swelling, tissue short-circuit current and bulk transporter measurements integrate responses from many epithelial cells. Stronger tests would combine selective deletion or activation of BEST4⁺ cells with live measurements of intracellular pH, calcium, cyclic GMP, bicarbonate flux and mucus expansion. These experiments could determine whether BEST4 itself is the principal anion pathway, whether it mainly amplifies CFTR-mediated secretion, or whether its greatest contribution is sensing and coordinating responses among neighboring cells. They could also clarify whether toxin-induced secretion requires the endogenous guanylin–uroguanylin circuit or is driven predominantly by pharmacological stimulation of GUCY2C.

Therapeutic development will depend on preserving the distinction between pathological hypersecretion and protective basal transport. GUCY2C signaling and bicarbonate movement may contribute to epithelial maintenance and mucus function even while excessive cyclic GMP or cyclic AMP drives diarrhea. This argues for approaches that limit abnormal signal amplitude, duration or cellular targeting instead of eliminating the pathway entirely. Cell-type-resolved physiology, supported by species with a conserved BEST4⁺ population, should help identify that therapeutic window and establish whether the proposed hub is a druggable controller or primarily a useful map of interacting intestinal transport mechanisms.

Subject of Research: BEST4⁺ intestinal epithelial cells and their role in ion transport and diarrheal mechanisms

Article Title: BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation

Article References: Qu, H.-Z., & Wang, X.-Q. (2026). BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation. Advanced Biotechnology, 4(3), Article 33. https://doi.org/10.1007/s44307-026-00126-7

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00126-7

Keywords: BEST4⁺ cells, intestinal epithelium, ion transport, CFTR, GC-C signaling, secretory diarrhea, viral gastroenteritis, mucus barrier, BEST4, cells, potential, intestinal