Keloids are chronic fibroproliferative lesions marked by persistent inflammation, altered wound healing and excessive extracellular matrix deposition. Unlike ordinary scars, they continue to enlarge beyond the original wound margins and may cause itching, pain and substantial cosmetic distress. Surgery, intralesional corticosteroids, radiotherapy, laser therapy and cryotherapy can reduce lesion burden in selected patients, yet recurrence is common. The study’s focus was therefore not a clinical treatment trial, but an attempt to connect inherited variation in circulating microRNAs with molecular changes measured in established keloid tissue and primary cells.

For the genetic component, the investigators used two-sample Mendelian randomization. Genetic variants associated with plasma microRNA levels were treated as instrumental variables for the corresponding circulating microRNAs, while summary association data from the Million Veteran Program were used to assess keloid susceptibility. The miRNA quantitative-trait-locus data came from a genome-wide association study of 2,178 participants in which 2,083 plasma microRNAs were quantified. The keloid outcome analysis included 447,008 analyzable individuals. Linkage-disequilibrium clumping, removal of palindromic variants and instrument-strength checks were used to prepare the analyses, with instruments for most microRNAs represented by a single genetic variant.

Nine microRNAs showed nominal associations with keloid susceptibility, but none remained significant after correction for multiple testing. miR-539-3p had a nominal inverse genetic association, with an odds ratio of 0.68, a 95% confidence interval of 0.47 to 0.97 and a P value of 0.035; its false-discovery-rate-adjusted P value was 0.986. Because that estimate came from one SNP, the investigators could not perform candidate-specific sensitivity analyses such as MR-Egger regression, weighted median estimation or leave-one-out analysis. The result is therefore an exploratory selection signal, not evidence that higher circulating miR-539-3p protects against keloids.

The researchers next analyzed the public GEO dataset GSE237753, which contains bulk microRNA sequencing from three keloid tissues and three normal skin controls. After quality control and filtering of low-abundance microRNAs, the DESeq2 analysis identified 44 differentially expressed microRNAs. miR-539-3p was the only candidate to overlap with the nominal Mendelian-randomization results. It was strongly upregulated in keloid tissue, with a log2 fold change of 3.29 and an adjusted P value of 4.71 × 10−6. This overlap was used to prioritize the molecule for biological follow-up rather than to confirm a genetic causal effect.

Potential downstream targets were assembled from miRDB, TargetScan and miRTarBase. Nine genes appeared in all three resources: SOX9, CTBP1, NRF1, ARID1A, SRSF2, PCGF3, HS3ST3B1, KPNA2 and MTF2. Their combined annotation was associated with developmental processes, regulation of cell number, stem-cell population maintenance and extracellular matrix organization, along with transcriptional and PI3K–Akt-related functions. SOX9 was selected for further examination because it was a shared predicted target, had a curated human miR-539-3p/SOX9 record in miRTarBase, and was considered biologically relevant to mesenchymal remodeling and fibrosis. The selection combined computational prediction, curated evidence and literature-guided biological judgment.

Experimental measurements in primary cells and tissue provided further support for the expression pattern. The researchers tested eight matched pairs of normal skin-derived and keloid-derived fibroblasts from eight patients. Both miR-539-3p and SOX9 messenger RNA levels were higher in the keloid-derived cells, with P values of 3.69 × 10−5 and 1.09 × 10−5, respectively. In an independent set of 20 specimens—10 normal skin samples and 10 keloids—semiquantitative immunohistochemistry showed stronger SOX9 staining in the dermal or lesional compartment of keloids. Mean immunoreactivity scores were 4.20 ± 1.38 for keloids and 1.55 ± 0.64 for normal skin, with P = 5.18 × 10−5.

SOX9-centered networks placed the candidate within several processes relevant to scar remodeling. GeneMANIA analysis connected SOX9 with genes involved in connective-tissue development, extracellular matrix organization and mesenchyme development, with enrichment for Notch signaling, cytoskeletal processes and PI3K–Akt signaling. A high-confidence STRING network was enriched for connective-tissue and mesenchyme development, epithelial-to-mesenchymal transition, collagen-containing extracellular matrix, Wnt signaling, TGF-β signaling, Hippo signaling, Hedgehog signaling and Notch signaling. These computational associations describe the biological context around SOX9; they do not demonstrate that SOX9 controls keloid formation or directly governs matrix deposition.

The investigators also transfected primary fibroblasts with a miR-539-3p inhibitor or a negative-control oligonucleotide. Across three independent experiments per assay, SOX9 messenger RNA was higher after inhibitor treatment than after control treatment, with P = 0.0007. Western blotting likewise showed higher SOX9-to-GAPDH protein abundance after inhibition, with a nominal P value of 0.0296. The result is compatible with the possibility that miR-539-3p can influence SOX9, but direct targeting was not tested. The study did not examine whether miR-539-3p binds the SOX9 3′ untranslated region, and it did not manipulate SOX9 to determine whether the protein changes fibroblast activation or matrix production.

Several limitations constrain the conclusions. The genetic signal was nominal, based on one SNP and unable to support robust candidate-specific sensitivity testing. The microRNA-QTL exposure cohort was of European ancestry, while the multi-ethnic keloid GWAS was predominantly European, limiting generalizability. The discovery transcriptomic dataset contained only three keloid and three control tissues, and the paired-cell and tissue experiments, although independent of that dataset, assessed established lesions rather than the process of scar initiation. The differing directions of the genetic and expression findings also reflect different biological contexts: genetically predicted circulating microRNA levels were compared with molecular measurements from established lesions and lesion-derived cells. The study has therefore narrowed a broad candidate space to an experimentally tractable miR-539-3p/SOX9 association. Direct binding assays, targeted perturbation of both molecules and rescue experiments in keloid-derived cells will be needed to determine whether the relationship is direct, indirect or simply part of a broader remodeling program.

Subject of Research: Exploratory genetic, transcriptomic and experimental analysis of miR-539-3p and SOX9 expression in keloid tissue and fibroblasts

Article Title: Integrated genetic and transcriptomic analyses suggest a miR-539-3p/SOX9-associated expression pattern in keloids

Article References: Luo, Z., Ge, L., Chen, L., Lin, S., Wei, T., Lu, B., & Jin, Z. (2026). Integrated genetic and transcriptomic analyses suggest a miR-539-3p/SOX9-associated expression pattern in keloids. Frontiers in Cell and Developmental Biology, 14, Article 1931861. https://doi.org/10.3389/fcell.2026.1931861

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1931861

Keywords: keloids, fibrosis, miR-539-3p, SOX9, microRNA, Mendelian randomization, transcriptomics, extracellular matrix, primary fibroblasts, Integrated, genetic, transcriptomic

The central challenge the team set out to address is a familiar one in regenerative medicine. Stem cells delivered into a defect are often metabolically unprepared for the hostile environment they encounter, remaining incompletely committed to the correct lineage and vulnerable to inflammatory and mechanical stress. Cartilage repair is especially unforgiving, because success requires not just cell survival but stable activation of a chondrogenic program, rich deposition of type II collagen and aggrecan, and active suppression of the fibrotic, hypertrophic and matrix-degrading pathways that cause engineered tissue to fail. Conventional approaches rely on biochemical cues such as transforming growth factor-β, which can switch on chondrogenic genes but provide little control over the intracellular metabolic state that must sustain durable matrix production over time.

The researchers’ strategy targets mitochondria, which have increasingly been recognized not as passive power plants but as active arbiters of stem-cell destiny. Mitochondrial membrane potential, respiratory activity, ATP availability and redox tone collectively determine whether a stem cell is merely alive or genuinely competent to regenerate tissue. Red light around 660 nanometers is absorbed by mitochondrial photoacceptors, most notably cytochrome c oxidase, also known as respiratory complex IV, linking photon input directly to electron transport, ATP synthesis and redox signaling. Unlike soluble biochemical factors, light can be applied externally, withdrawn instantly and spatially controlled, making it an attractive tool for preconditioning cells before they are ever placed inside a patient.

To find the right dose, the team systematically tested 5, 15 and 30 joules per square centimeter of 660-nanometer LED light on rabbit BMSCs, using a power density of 50 milliwatts per square centimeter and carefully monitoring culture temperature with infrared thermal imaging to rule out nonspecific heating. The results revealed a classic biphasic dose-response. At 15 joules per square centimeter, the cells proliferated most vigorously, showed increased S-phase entry by flow cytometry, and maintained high viability. The lowest dose produced only a modest effect, while the highest dose failed to enhance proliferation further and actually reduced cellular activity. Notably, a temperature-matched control group that experienced the same warming without light did not reproduce the inhibition seen at the high dose, indicating that the effect was photobiological rather than thermal.

The critical question was whether this proliferative boost translated into genuine chondrogenic commitment. When BMSCs were formed into cartilage pellets and irradiated daily for 14 days, the 15-joule group produced pellets that were larger, more regular and more compact than any other group. Histological staining showed denser tissue organization and stronger proteoglycan deposition, and biochemical assays confirmed elevated total collagen, type II collagen and glycosaminoglycan content. Crucially, when matrix production was normalized to DNA content, the ratios remained highest in the irradiated group, demonstrating that light enhanced the biosynthetic output of each cell rather than simply expanding the cell population. Western blotting revealed increased SOX9, type II collagen and aggrecan, while the unwanted markers COL1A1, COL10A1, RUNX2 and MMP13, which signal fibrosis, hypertrophy and matrix degradation, were all reduced at both protein and mRNA levels.

The mechanistic heart of the study lies in its dissection of the mitochondrial signaling cascade. At the optimal dose, LED irradiation elevated mitochondrial membrane potential, produced a more interconnected mitochondrial network, increased ATP production and enhanced respiratory parameters including basal respiration, ATP-linked respiration, maximal respiration and spare respiratory capacity, as measured by Seahorse extracellular flux analysis. Cytochrome c oxidase activity was also increased. Perhaps most intriguingly, the treatment induced a moderate rise in mitochondrial hydrogen peroxide, a controlled burst of reactive oxygen species that appears to function as a signaling mediator rather than a damaging by-product. At 30 joules per square centimeter, by contrast, ROS accumulation became excessive, accompanied by elevated malondialdehyde and 4-hydroxynonenal, hallmarks of oxidative stress and lipid peroxidation.

Using a panel of pharmacological inhibitors, the researchers traced the pathway from mitochondria to the nucleus. Scavenging ROS with N-acetyl-L-cysteine attenuated the phosphorylation of PI3K, Akt, mTOR and GSK3β, placing moderate mitochondrial ROS upstream of the cascade. Inhibiting PI3K with LY294002 or Akt with MK-2206 suppressed downstream phosphorylation and reduced SOX9, type II collagen and aggrecan expression, while the mTOR inhibitor rapamycin blunted glycosaminoglycan deposition. The enhanced inhibitory phosphorylation of GSK3β, which reduces its negative regulatory activity, further supported SOX9 nuclear activation, confirmed by immunofluorescence showing stronger nuclear SOX9 signal in irradiated cells. Together, these experiments establish the PI3K/Akt/mTOR/GSK3β–SOX9 axis as the conduit through which photonic energy is converted into a chondrogenic transcriptional program.

The team then translated the optimized treatment into a tissue-engineering construct. Egg white-derived biomaterial was fabricated into ring-shaped porous scaffolds, seeded with BMSCs and irradiated for 14 days before implantation into a rabbit tracheal defect created by resecting one native cartilage ring. At 30 days after surgery, bronchoscopic evaluation showed higher airway patency, reduced granulation formation, lower stenosis and less collapse in the 15-joule group compared with untreated and high-dose controls. Histology revealed organized, proteoglycan-rich cartilage-like matrix, and biochemical analysis showed markedly increased GAG/DNA and COL II/DNA levels that approached, though did not fully reach, those of native tracheal tissue. Fibrotic, hypertrophic and matrix-degrading markers were suppressed in vivo, and epithelial repair was improved, with restored basal coverage and ciliated differentiation alongside reduced injury-associated mucus-producing remodeling.

Most compellingly, direct mechanical benchmarking against native trachea showed that the reconstructed rings in the 15-joule group exhibited higher radial compression modulus, greater anti-collapse pressure, improved bending recovery and lower residual deformation than the other groups, with all four parameters shifting toward the native range. The trachea depends on its cartilage rings to resist collapse during every breath, so restoring load-bearing function is the ultimate test of any repair strategy. The irradiated constructs also resisted osteogenic drift, showing no increase in osteocalcin at the optimized dose, while the high-dose group did, reinforcing the importance of precise photonic dosing. The researchers caution that the 30-day observation period was short, that a temperature-matched control specifically matching the 15-joule condition was not included, and that genetic approaches would further strengthen causal interpretation of the signaling pathway.

What distinguishes this work from earlier photobiomodulation studies is its conceptual shift. Previous efforts applied light directly to injured tissues to modulate the local repair environment; here, light is used before implantation to write a regenerative metabolic state into the cells themselves. The 15-joule condition represents a functional window that coordinates mitochondrial activation with cellular adaptation, highlighting that the goal is not maximal ROS production but a favorable bioenergetic-redox balance. The treatment also showed immunomodulatory promise, shifting macrophages from an inflammatory M1-like phenotype toward a reparative M2-like state and preserving chondrogenic gene expression under interleukin-1β challenge, a scenario that mirrors the inflammatory milieu of real wounds. If the approach can be adapted to larger animal models and clinically relevant reconstruction scenarios, externally controlled, growth factor-sparing photonic preconditioning could become a practical and elegant tool for engineering cartilage that not only fills a defect but performs the mechanical work the body demands of it.

Subject of Research: 660 nm LED mitochondrial preconditioning of bone marrow-derived mesenchymal stem cells for functional tracheal cartilage regeneration

Article Title: 660 nm LED preconditioning programs mitochondrial signaling in BMSCs for functional tracheal cartilage regeneration

Article References: An, Z., Zhang, J., Wang, C., She, Y., Xu, Y., Zhou, H., Xia, J., Guo, L., Ma, M., & Zhang, L. (2026). 660 nm LED preconditioning programs mitochondrial signaling in BMSCs for functional tracheal cartilage regeneration. Materials Today Bio, 41, Article 103729. https://doi.org/10.1016/j.mtbio.2026.103729

Image Credits: AI Generated

DOI: 10.1016/j.mtbio.2026.103729

Keywords: photobiomodulation, 660 nm LED, mesenchymal stem cells, mitochondria, chondrogenesis, tracheal cartilage, tissue engineering, SOX9, PI3K/Akt/mTOR signaling, reactive oxygen species, cytochrome c oxidase, scaffold

The central player in the study is SOX9, short for SRY-box transcription factor 9. Transcription factors are proteins that bind to specific DNA sequences and control whether nearby genes are read into messenger RNA, and SOX9 is well known for its roles in embryonic development and tissue stem cell maintenance. In cancer, SOX9 has previously been implicated in tumor growth and progression, but its involvement in the metabolic transformation of metastatic colorectal cancer cells had remained largely unexplored. The new work positions SOX9 as a direct regulator of de novo lipogenesis, the process by which cells manufacture fatty acids from scratch rather than importing them from the bloodstream.

To dissect this relationship, the researchers combined several complementary approaches. They performed integrated transcriptomic and targeted lipidomic analyses, comparing the gene expression profiles and lipid compositions of cells with and without elevated SOX9. The transcriptomic data revealed changes in the expression of genes involved in fatty acid synthesis, while the lipidomic measurements showed that SOX9 overexpression increased the abundance of fatty acids and other lipid species within the cells. This convergence of genomic and metabolomic evidence provided the first indication that SOX9 overexpression drives fatty acid synthesis and lipid accumulation in colorectal cancer cells.

The team then verified these findings using an arsenal of imaging and analytical techniques. Nile Red staining, a fluorescent dye that preferentially labels neutral lipids, revealed increased lipid content in SOX9-overexpressing cells. Flow cytometry allowed the researchers to quantify lipid accumulation across large cell populations, confirming that the effect was consistent rather than confined to a few cells. Transmission electron microscopy provided ultrastructural evidence, visualizing the intracellular lipid deposits at nanometer resolution. Finally, Nanolive label-free lipid droplet imaging captured the dynamics of lipid storage structures inside living cells. Together, these methods demonstrated that SOX9 overexpression dramatically enhances intracellular lipid deposition, effectively transforming colorectal cancer cells into lipid-storing factories.

The clinical relevance of these observations became apparent when the researchers examined human tissue samples. SOX9 was found to be upregulated in colorectal cancer tissues compared with healthy tissue, and its expression was further enriched in metastatic lesions, the colonies of cancer cells that have established themselves in distant organs. Importantly, patients whose tumors showed high SOX9 levels had poorer outcomes, suggesting that the transcription factor is not merely a bystander in disease progression but a marker, and likely a driver, of aggressive disease.

Functional experiments cemented the link between SOX9-driven lipid production and metastatic behavior. In laboratory assays, cells with elevated SOX9 showed markedly increased migration and invasion, the two cellular behaviors that precede and enable metastatic spread. The team also performed lung metastatic colonization assays in animal models, which measure the ability of circulating cancer cells to seed and grow tumors in the lung. SOX9 overexpression promoted lung metastatic colonization, demonstrating that the lipid-reprogramming effect translates into real metastatic capacity in vivo.

Perhaps the most therapeutically significant result came from the rescue experiments. When the researchers inhibited fatty acid synthase, commonly abbreviated FASN, the enzyme that catalyzes the final steps of fatty acid synthesis, the pro-metastatic effects of SOX9 were significantly attenuated. Migration, invasion and lung colonization all declined when the fat-production line was shut down, even in cells still overexpressing SOX9. This result indicates that FASN sits downstream of SOX9 in the pathway and that the lipid synthesis it drives is not incidental but functionally required for SOX9 to promote metastasis. In principle, this makes the SOX9-FASN axis a metabolic vulnerability that could be targeted with drugs designed to starve metastatic cells of their lipid supply.

Mechanistically, the study went beyond correlation to show how SOX9 controls FASN in the first place. Using CUT&Tag sequencing, a technique that maps where a protein of interest binds across the genome, the researchers identified SOX9 occupancy at sites near the FASN promoter, the regulatory DNA region that controls how strongly the FASN gene is transcribed. This genomic binding evidence was complemented by luciferase reporter assays, in which the FASN promoter was fused to a light-producing reporter gene. When SOX9 was present, reporter activity increased, demonstrating that SOX9 can directly stimulate FASN promoter activity. Further reporter analyses using mutated promoter sequences supported a sequence-dependent mode of regulation, meaning SOX9 requires specific DNA motifs in the proximal FASN promoter to exert its effect, a hallmark of genuine direct transcriptional control rather than an indirect effect mediated through other genes.

The significance of this work lies in how it connects two major themes in modern cancer biology: transcriptional dysregulation and metabolic reprogramming. Cancer cells are known to rewire their metabolism to support rapid growth, survival under stress and dissemination, and increased lipid synthesis has emerged as a recurring feature of aggressive tumors. Lipids are not merely building blocks for new membranes in dividing cells; they also serve as signaling molecules, energy reserves and components of the machinery cells use to change shape and move through tissue. By showing that a developmental transcription factor can impose a lipogenic program on colorectal cancer cells and that this program is required for metastatic colonization, the study offers a coherent explanation for how metabolic flexibility and metastatic competence are coupled at the level of gene regulation.

The findings also suggest practical directions for future research and treatment development. Because FASN inhibition can rescue the metastatic phenotypes induced by SOX9, drugs targeting fatty acid synthase, some of which have already entered clinical evaluation for other cancers, could potentially benefit colorectal cancer patients whose tumors show high SOX9 expression. SOX9 itself, as a transcription factor, has historically been considered a difficult drug target, but its downstream metabolic dependencies offer an alternative point of attack. The study also raises the possibility that SOX9 expression levels could serve as a biomarker to identify patients at high risk of metastasis who might be candidates for metabolic therapies. As with all preclinical research, the results will need to be validated in larger clinical cohorts and tested in appropriately designed trials before they change patient care, but the identification of a defined SOX9-FASN lipid metabolic axis gives researchers a concrete molecular handle on one of the deadliest phases of colorectal cancer.

Subject of Research: SOX9-mediated regulation of lipid metabolism and metastasis in colorectal cancer

Article Title: SOX9 reprograms lipid metabolism via FASN to promote metastasis in colorectal cancer

Article References: Wang, H., Wei, L., Xiang, X., Li, J., Wang, Y., Jiang, Z., Li, X., Li, Y., Pan, Y., & Liang, X. (2026). SOX9 reprograms lipid metabolism via FASN to promote metastasis in colorectal cancer. Journal of Translational Medicine. https://doi.org/10.1186/s12967-026-09055-4

Image Credits: AI Generated

DOI: 10.1186/s12967-026-09055-4

Keywords: colorectal cancer, SOX9, FASN, lipid metabolism, metastasis, fatty acid synthesis, de novo lipogenesis, transcription factors, cancer metabolism, CUT&Tag, lung metastasis, Journal of Translational Medicine

The study, published in BMC Genomics by a research group led by Xinxin Cao, Lihui Zhang, Kaidong Liu, Jinshan Zhao and Hegang Li, focused on dairy goat embryos at 55 to 65 days of gestation, a critical window during which the gonads commit to their developmental trajectories. The team analyzed three groups of embryos: genetic females, genetic males, and intersex fetuses affected by polled intersex syndrome. Using single-cell RNA sequencing, the researchers profiled the gene expression of thousands of individual cells, then combined this with tissue morphology and immunofluorescence staining to confirm where key proteins were actually located within the developing organs.

Single-cell RNA sequencing works by dissociating a tissue into its component cells and capturing the messenger RNA from each one, allowing researchers to see which genes are active in which cells rather than averaging signals across a whole organ. In this study, that approach revealed striking cellular heterogeneity within the embryonic gonads. The team identified distinct populations of germ cells, the precursors of eggs and sperm, alongside a diverse cast of somatic cells, including stromal cells, granulosa cells, endothelial cells, immune cells, and, in males, Sertoli cells, interstitial cells and epithelial cells. The composition and proportions of these populations differed dramatically between female, male and intersex gonads.

One of the most consequential findings concerns where the different gonadal lineages come from. Through computational reconstruction of differentiation trajectories, a technique known as pseudotime analysis that orders cells along a developmental path based on their gene expression, the researchers found evidence that stromal cells may initiate the development of female and intersex gonads, while epithelial cells appear to serve as the origin of male gonadal cells. This suggests that the cellular ancestry of a gonad may be set remarkably early, and that in intersex embryos the developmental program may be steered toward a male-like trajectory from its starting point.

The molecular culprits behind sex reversal are familiar names in developmental biology, but the study adds new nuance. The researchers documented aberrant expression of SOX9, AMH and DMRT1, a trio of genes that together drive testis formation. SOX9 is the master switch of testis development; AMH, produced by Sertoli cells, triggers the regression of female reproductive structures; and DMRT1 reinforces the male program. In the intersex gonads, these genes were misexpressed in ways consistent with a partial female-to-male conversion. The finding confirms that the same core genetic circuitry implicated in disorders of sex development in humans is also at work in goats with polled intersex syndrome.

Perhaps the most exciting aspect of the study is the identification of new candidate regulators. The researchers flagged PHLDB2, ZNRF3 and TEAD1 as novel genes potentially involved in orchestrating sex reversal. ZNRF3 is known from other contexts to participate in Wnt signaling regulation, a pathway with well-established roles in ovary determination, while TEAD1 sits at the heart of the Hippo signaling pathway, which governs organ size and cell fate. Their emergence from an unbiased single-cell screen suggests that the network controlling gonadal sex determination is even more intricate than the canonical SOX9-centered model implies, and opens new avenues for experimental validation.

The study also delivered a surprise from the immune compartment. The team detected TREM2-positive macrophages within the goat embryonic gonads, a cell population that had not previously been characterized in this setting. Macrophages are best known as immune scavengers, but growing evidence implicates them in shaping tissue environments during organogenesis. The researchers propose that multiple genes expressed in these and other gonadal cells may modulate sex reversal by altering the gonadal microenvironment, the local signaling milieu in which germ cells and somatic cells make their fate decisions. In other words, sex determination may not be a purely cell-autonomous process but one influenced by the surrounding cellular ecosystem.

In a finding that borders on the unexpected, the analysis also implicated circadian rhythm-related genes in early gonadal development and the formation of intersex traits. Circadian genes are classically associated with daily biological rhythms, yet they have increasingly been shown to influence developmental timing in various tissues. Their differential expression across female, male and intersex gonads hints that the internal clock machinery may intersect with the sex determination network, a connection that, if confirmed experimentally, would add an entirely new dimension to how scientists think about disorders of sexual development.

The practical implications for livestock breeding are immediate. Polled intersex syndrome arises in goats bred for the polled, or hornless, trait, because the genetic mutation responsible for hornlessness is tightly linked to the intersex condition. Understanding precisely which cells and genes go awry during early gonad formation could enable breeders to select against the syndrome more effectively, protecting productivity in dairy herds. The single-cell atlas generated by this study provides a molecular reference that could support marker-assisted selection or even gene-editing strategies aimed at decoupling the polled trait from intersex development.

But the study’s ambitions extend well beyond the barnyard. The authors emphasize that polled intersex syndrome in goats serves as a valuable natural model for human reproductive defects, including disorders of sex development that affect roughly one in thousands of newborns worldwide. Because the goat gonad develops through mechanisms broadly comparable to those in humans, the cellular and molecular pathways mapped here, from stromal and epithelial origins to SOX9, AMH and DMRT1 dysregulation, from immune modulation to circadian influences, offer a framework for investigating why and how human sex development sometimes diverges. As single-cell technologies continue to mature, studies like this one demonstrate that even a familiar farm animal can illuminate some of the most fundamental and intimate questions in human biology: how a tiny embryonic organ decides what it will become, and what happens when that decision goes awry.

Subject of Research: Single-cell transcriptomic analysis of intersex gonad initiation and early sex differentiation in dairy goat embryos

Article Title: Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats

Article References: Cao, X., Zhang, L., Liu, K., Zhang, Y., Liu, X., Xu, D., Lin, X., Campo, F. P., Sun, M., Qin, Z., Gao, X., He, J., Zhao, J., & Li, H. (2026). Single-cell RNA sequencing analysis reveals new insights for intersex gonad initiation and early sex differentiation of dairy goats. BMC Genomics. https://doi.org/10.1186/s12864-026-13404-4

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13404-4

Keywords: single-cell RNA sequencing, polled intersex syndrome, dairy goats, sex differentiation, gonad development, SOX9, AMH, DMRT1, pseudotime analysis, germ cells, macrophages, circadian rhythm genes

The research team, led by Matthias Weider and Lina Gölz of University Hospital Erlangen together with colleagues in Bonn, began by making IRF6 visible in developing mouse embryos. Using transgenic animals in which all neural crest-derived tissue glows yellow thanks to a Wnt1::Cre-driven fluorescent reporter, the scientists stained embryonic sections at days 9.5, 10.5 and 11.5 of development. They found IRF6 protein in neural crest-derived cells of the pharyngeal arches, the embryonic structures that give rise to the jaw, palate and other facial components. Intriguingly, the fraction of neural crest cells carrying high levels of IRF6 shrank as development progressed: by embryonic day 11.5, only about 2.6 percent of these cells retained signal intensity comparable to that of epithelial cells, hinting at a narrow, precisely timed window of activity.

To confirm that this was not a quirk of the mouse or of the staining method, the team mined single-cell RNA sequencing data from embryonic craniofacial tissue of both mouse and human origin. The computational analysis revealed a subset of mesenchymal cells expressing IRF6 in both species. Crucially, many of these IRF6-positive cells also expressed SOX9, with 56 percent of IRF6-positive mesenchymal cells co-expressing the gene, and an even larger share, 86.8 percent, co-expressing TFAP2A, the transcription factor already known to regulate IRF6 in epithelium. The overlap placed IRF6 squarely inside the regulatory world of cranial neural crest cells, a transient, stem cell-like population whose migration, proliferation and differentiation must be choreographed with extraordinary precision for the face to form correctly.

SOX9 was a compelling suspect for several reasons. It is a bona fide cranial neural crest transcription factor, essential for assigning cells to the cartilage lineage, and it is the major gene mutated in Pierre Robin sequence, a craniofacial condition characterized by an underdeveloped lower jaw, a tongue that falls back into the throat, and frequently a cleft palate. Moreover, a single nucleotide variant called rs76145088, which genetic studies had previously linked to non-syndromic orofacial clefting, sits within a DNA sequence in the IRF6 enhancer MCS-9.7 that resembles a SOX9 binding site. That enhancer, located thousands of base pairs upstream of IRF6, was already famous: a different variant within it, rs642961, disrupts binding of TFAP2A and increases cleft risk, helping to establish MCS-9.7 as a hotspot of cleft-associated regulatory variation.

To test whether SOX9 really drives the enhancer, the researchers cloned MCS-9.7 into a luciferase reporter plasmid, a standard tool that glows when the enhancer is active. In neural crest-derived Neuro-2a cells, they first reproduced the known activation by TFAP2A, validating the system. They then added SOX9 and found that it, too, switched the enhancer on. The striking result concerned the rs76145088 variant: the ancestral allele, which is the risk allele for orofacial clefting, supported robust SOX9-dependent activation, while the derived allele, whose guanine base breaks the SOX9 consensus sequence, drove drastically weaker activation. Dose experiments sharpened the point, as 250 nanograms of SOX9 expression plasmid sufficed to activate the ancestral version of the enhancer, whereas three times that amount was needed for the derived version.

The team then moved from artificial reporters to endogenous biology. In O9-1 cells, a stable cell line derived from mouse cranial neural crest, they silenced Sox9 with short hairpin RNAs and watched the activity of the ancestral enhancer allele drop, confirming that normal cellular levels of Sox9 sustain MCS-9.7 activity. Electrophoretic mobility shift assays showed that purified SOX9 protein binds directly to the DNA sequence spanning rs76145088, and that it binds the ancestral allele more strongly than the derived one, exactly as the reporter experiments predicted. Chromatin immunoprecipitation in O9-1 cells, pulling down DNA bound by Sox9 inside living cells, enriched the region corresponding to MCS-9.7 relative to a negative control, albeit modestly, consistent with an enhancer that is active in only a small subset of cells.

The decisive experiment came next. Using CRISPR/Cas9 gene editing, the researchers knocked out Sox9 in O9-1 cells, generating two independent clones in which the protein was completely absent, one through a frameshift mutation and the other through a dramatic rearrangement including an inversion around the start codon. Quantitative PCR revealed that Irf6 transcript levels were virtually eliminated in both knockout clones. Other known upstream regulators of Irf6, including Tfap2a, Notch1, Foxe1, Trp63, the Pbx genes and several Wnt genes, showed only mild and inconsistent changes, arguing that SOX9 acts directly on the IRF6 enhancer rather than through intermediate regulators. Notably, the well-established IRF6 target gene Grhl3 was not expressed in these neural crest cells, suggesting that IRF6 may control a different set of downstream genes in cranial neural crest cells than it does in epithelium.

Perhaps the most thought-provoking twist is the direction of the risk effect. Usually, loss-of-function mutations in IRF6 cause clefting by reducing gene activity in epithelium. Here, the ancestral, risk-associated allele of rs76145088 is the one that binds SOX9 more strongly and drives higher enhancer activity. The authors therefore propose a gain-of-function mechanism: prolonged or excessive IRF6 expression in craniofacial mesenchyme could contribute to clefting, in the same way that enhanced binding of the transcription factor ETS1 to an SHH enhancer causes polydactyly. Because rs76145088 is common across human populations while clefts remain relatively rare, the finding fits the multifactorial threshold model of non-syndromic clefting, in which risk accumulates from many variants and environmental influences before a developmental threshold is crossed. The authors also note that the derived allele may be protective rather than the ancestral allele harmful, and that transcription factors like IRF6 must be kept within tight dosage windows, since both too little and too much activity can derail embryonic development.

The study does not close the case. Only a small fraction of cranial neural crest cells express IRF6, the enhancer carries both active and repressive histone marks suggestive of a poised or heterogeneous state, and previous mouse experiments deleting Irf6 in neural crest lineages produced bone mineralization defects but no palatal clefts. Still, by connecting a classic epithelial cleft gene to SOX9, the gene behind Pierre Robin sequence, the work draws two major cleft-associated pathways into a single regulatory axis and reveals that the developing face depends on IRF6 operating in more cellular neighborhoods than anyone had appreciated. Dysregulation of the SOX9-IRF6 axis in cranial neural crest cells, the authors conclude, may be a genuine contributor to the origins of orofacial clefting, and the heterogeneous mesenchymal cells that carry this signal are now an obvious target for the next generation of studies.

Subject of Research: Transcriptional regulation of the orofacial cleft risk gene IRF6 by SOX9 in cranial neural crest cells

Article Title: The orofacial cleft risk gene IRF6 is a target gene of SOX9 in cranial neural crest cells

Article References: Weider, M., Wagner, M. C., Schmid, T., Gehlen-Breitbach, S., Rodrian, G., Peschel, N., Schneider, H., Ludwig, K. U., Wegner, M., & Gölz, L. (2026). The orofacial cleft risk gene IRF6 is a target gene of SOX9 in cranial neural crest cells. Journal of Molecular Medicine, 104(1), Article 113. https://doi.org/10.1007/s00109-026-02720-7

Image Credits: AI Generated

DOI: 10.1007/s00109-026-02720-7

Keywords: orofacial cleft, IRF6, SOX9, cranial neural crest cells, cleft lip and palate, Pierre Robin sequence, MCS-9.7 enhancer, TFAP2A, CRISPR/Cas9, gene regulation, craniofacial development, single-cell RNA sequencing

The research builds on the group’s earlier work showing that myeloid-derived suppressor cells, or MDSCs, accumulate in abundance within human esophageal squamous cell carcinoma, the dominant histological subtype of esophageal cancer worldwide, and that their presence correlates with worse patient outcomes. What remained unclear was whether MDSCs contribute to tumor propagation through mechanisms that go beyond their canonical role of suppressing T cells. To answer that question, the investigators turned to mouse models of esophageal cancer that included both immunocompetent animals and mice lacking T cells altogether. If MDSCs promoted tumor growth only by disarming T cells, their influence should have vanished in the T cell-deficient setting. Instead, the tumors accelerated in both contexts, a result that signaled the existence of an intrinsic, T cell-independent tumor-promoting program driven by these myeloid cells.

Tracking down the molecular machinery behind that program led the team to an unexpected transcription factor. When the researchers depleted MDSCs in tumor-bearing mice using an anti-Gr1 antibody, or when they neutralized the inflammatory cytokine interleukin-1 beta, expression of HNF1A inside the tumor cells dropped. HNF1A, a transcription factor best known for its roles in liver and pancreatic development and metabolism, has more recently been implicated in tumor biology, but its involvement in MDSC-driven esophageal cancer progression had not been established. The new data place it at the center of the circuit: MDSCs, acting through IL-1 beta, appear to switch on HNF1A within the carcinoma cells themselves.

From HNF1A, the signal flows onward to a chemokine. The study showed that HNF1A sustains the activity of the CXCL1 promoter, and that disrupting the upstream inputs, whether by depleting MDSCs or blocking IL-1 beta, blunted CXCL1 promoter activity and reduced CXCL1 output. CXCL1 is a chemokine of the CXC family that signals through the receptor CXCR2 and is well recognized as a mediator of neutrophil recruitment and inflammatory signaling in tumors. In this context, however, its downstream consequences are not primarily inflammatory in the classical sense. Rather, the researchers found that the HNF1A-CXCL1 axis feeds directly into the core stemness machinery of the cancer cells, downregulating or, when the axis is active, sustaining the expression of the transcription factors Oct4, Nanog and Sox9, the canonical guardians of stem-like identity in embryonic and cancer stem cells alike.

The functional consequences of that molecular cascade were tested with rigor. When the team deleted HNF1A specifically in tumor cells, the MDSC-driven expansion of the cancer stem cell population collapsed, and, critically, the ability of the tumors to initiate new growth was abolished. Tumor initiation is the hallmark functional readout of cancer stem cell activity: only cells with genuine self-renewal capacity can seed a new tumor from a limited inoculum. The fact that HNF1A deletion eliminated this capacity demonstrates that the transcription factor is not merely a correlate of stemness but a required licensing factor in this pathway. Conversely, when the researchers supplied exogenous IL-1 beta, the cancer stem cell frequencies rebounded, confirming that the myeloid-cell-derived cytokine sits upstream of the entire axis and is sufficient to re-ignite the stem-like program.

These results reframe the relationship between inflammation and cancer stemness in esophageal cancer. Rather than acting as passive bystanders that merely modulate the immune microenvironment, MDSCs emerge as active instructors of tumor cell identity, delivering an IL-1 beta signal that is transcribed into a heritable, self-reinforcing stem-like state through HNF1A and CXCL1. Because the pathway was operative in T cell-deficient mice, the authors conclude that MDSC-derived IL-1 beta licenses the HNF1A-CXCL1 axis and sustains cancer stem cell properties independently of adaptive immunity. That independence matters clinically. Modern oncology has invested heavily in T cell-directed strategies, from immune checkpoint inhibitors to engineered cell therapies, and esophageal cancer has been among the tumor types to benefit. But a tumor-promoting program that runs beneath the T cell layer provides a reservoir of malignant potential that such therapies would not touch, and it may help explain why responses in esophageal squamous cell carcinoma remain incomplete for many patients.

The therapeutic implication drawn by the authors is that targeting this pathway could complement, rather than replace, T cell-focused treatments. Several points of intervention suggest themselves from the data. MDSC depletion, as achieved with anti-Gr1 in the preclinical setting, removes the source of the signal. IL-1 beta neutralization interrupts the messenger, and IL-1-blocking agents already exist in the clinical arsenal for inflammatory diseases, offering a plausible route to translation. Downstream, the CXCL1-CXCR2 axis is a recognized drug target, with CXCR2 antagonists under investigation in multiple cancers. Each of these strategies was supported, directly or indirectly, by the experimental results: depleting MDSCs or neutralizing IL-1 beta reduced HNF1A expression, dampened CXCL1 promoter activity and lowered stemness factor levels, while restoring IL-1 beta reinstated cancer stem cell frequencies.

The study also carries prognostic weight. The graphical summary accompanying the article emphasizes that the HNF1A-driven CXCL1 program not only licenses stem-like properties in esophageal squamous cell carcinoma but also predicts poor prognosis, consistent with the team’s earlier finding that MDSC abundance in human tumors correlates with adverse outcomes. Cancer stem cells are widely associated with resistance to chemotherapy and radiotherapy, metastatic dissemination and relapse after apparently curative treatment, so a microenvironmental signal that expands this compartment provides a mechanistic bridge between inflammatory infiltration and clinical aggressiveness. For patients with esophageal squamous cell carcinoma, a disease with persistently poor survival statistics in many regions, that bridge may represent one of the more actionable links identified to date.

Methodologically, the work combined genetically defined mouse models, cell-specific deletion of HNF1A, pharmacologic MDSC depletion, cytokine neutralization and supplementation, and molecular readouts of promoter activity and stemness factor expression, an integrated design that allowed the authors to move from correlation to causal mechanism. The study was supported by the National Natural Science Foundation of China, and the MEC25 cell line used in the experiments was provided by the laboratory of Professor Li Fu at Shenzhen University Medical School. The corresponding author, Yi Zhang, holds appointments across the Biotherapy Center and Cancer Center of the First Affiliated Hospital of Zhengzhou University, the State Key Laboratory of Metabolic Dysregulation and Prevention and Treatment of Esophageal Cancer, and related Zhengzhou University institutions, reflecting the translational infrastructure behind the project. The article was received in January 2026, accepted in September 2026 and published on 20 September 2026 under a Creative Commons Attribution license.

As with any preclinical study, the path from mouse models to patient benefit will require validation in human tumor specimens and, ultimately, clinical testing of pathway-targeted interventions. Nevertheless, the conceptual advance is clear and consequential: the immune microenvironment does not merely decide whether the immune system sees a tumor, it can also decide what the tumor is. In esophageal squamous cell carcinoma, myeloid-derived suppressor cells appear to whisper a developmental command into the cancer cells, activating HNF1A, broadcasting CXCL1 and preserving a self-renewing core that survives whatever the immune system or the oncologist throws at it. Silencing that command, whether by removing the myeloid messengers, intercepting their IL-1 beta message or blocking the CXCL1 relay downstream, now stands as a defined and testable strategy to strip esophageal cancer of its stem-like resilience and to make T cell-directed therapies work against a smaller, more vulnerable target.

Subject of Research: How myeloid-derived suppressor cells confer cancer stem cell properties to esophageal squamous cell carcinoma via the HNF1A/CXCL1 signaling axis

Article Title: Myeloid-derived suppressor cells confer stemness to esophageal cancer cells through the HNF1A/CXCL1 signaling axis

Article References: Qin, G., Ma, P., Liu, S., Chen, T., Guo, K., Zhao, Q., Wu, P., Chen, X., & Zhang, Y. (2026). Myeloid-derived suppressor cells confer stemness to esophageal cancer cells through the HNF1A/CXCL1 signaling axis. Cancer Immunology, Immunotherapy. https://doi.org/10.1007/s00262-026-04577-8

Image Credits: AI Generated

DOI: 10.1007/s00262-026-04577-8

Keywords: myeloid-derived suppressor cells, esophageal squamous cell carcinoma, HNF1A, CXCL1, cancer stem cells, interleukin-1 beta, tumor microenvironment, stemness, Oct4, Nanog, Sox9, cancer immunology

The research team, led by investigators at the First Affiliated Hospital of Xinjiang Medical University, combined single-cell RNA sequencing, spatial transcriptomics, CRISPR-based gene editing, pharmacological pathway modulation, and xenograft modeling to trace SOX9’s role across both time and tissue space. Using a publicly available single-cell dataset, they reconstructed a pseudotime trajectory with Monocle3, mapping the continuous transition from astrocytes to malignant glioma cells. The analysis revealed substantial cellular heterogeneity within glioma samples, encompassing malignant cells alongside astrocytes, macrophages, T cells, B cells, monocytes, neurons, fibroblasts, and endothelial cells.

The most consequential finding to emerge from the trajectory analysis was SOX9’s temporal behavior. Its expression was concentrated almost exclusively in astrocyte and malignant cell populations, and along the inferred developmental arc it peaked early and then declined steadily as cells matured into malignant states. Correlation analysis confirmed this downward trend, with Pearson and Spearman coefficients both strongly negative and highly significant. At the level of trajectory nodes, SOX9 dominated the early, astrocyte-rich segments and faded in the terminal malignant zones, a pattern inconsistent with the conventional view of SOX9 as a constitutively active stemness factor.

To probe what SOX9 might be doing during that early window, the researchers scored the activity of the Hippo pathway, a master regulator of organ size and stem cell fate whose downstream effectors YAP and TAZ are established drivers of glioblastoma plasticity. Using Gene Set Variation Analysis, they quantified an upstream kinase module, including MST1/2, LATS1/2, SAV1, MOB1A/B, NF2, and WWC1, and a canonical YAP/TAZ target module containing genes such as CTGF, CYR61, ANKRD1, AXL, and BIRC5. Both modules showed inverse relationships with SOX9 expression, and generalized additive modeling revealed that pseudotime and SOX9 each contributed independent, nonlinear effects on pathway activity. In other words, the SOX9–Hippo relationship was phase-dependent, strongest during early-to-intermediate stages of the transition and not a simple monotonic association.

Spatial transcriptomics added a second, geographic dimension to the story. Analyzing four anatomically distinct regions of glioblastoma tissue—the tumor–normal interface, the pure tumor core, the perivascular compartment, and the tumor–necrosis interface—the team mapped SOX9 expression against the probability that each spatial spot contained malignant cells. The coupling between SOX9 and malignancy was weak or unstable at the tumor edges and the necrotic margin, modest in the tumor core, and most robust in the perivascular niche. There, SOX9 expression was markedly elevated, correlations with malignant cell probability were strongest, and neighborhood enrichment and spatial autocorrelation statistics all confirmed significant co-localization.

This regional specificity is biologically meaningful. The perivascular niche has long been recognized as a reservoir for stem-like glioblastoma cells, bathed in vascular, hypoxic, and paracrine signals that nurture cellular plasticity. The findings suggest that SOX9-dependent reprogramming is not only time-restricted but niche-conditioned, with perivascular regions providing the most permissive anatomical context for effective SOX9–Hippo–YAP/TAZ coupling. Elsewhere in the tumor, downstream malignant programs may be sustained through alternative inputs, weakening the spatial coherence of the axis.

Functional experiments brought the correlation studies into the laboratory. Using lentiviral vectors, the team generated U87 glioma cells stably overexpressing SOX9 and used CRISPR/Cas9 to knock out the gene in U251 cells, validating the edits by Sanger sequencing and confirming a frameshift-inducing deletion in the knockout clone. SOX9 overexpression modestly increased proliferation, migration, and invasion while reducing apoptosis, whereas SOX9 knockout produced the opposite phenotype across wound-healing, Transwell invasion, and flow-cytometric apoptosis assays. Critically, treatment with XMU-MP-1, an inhibitor of the upstream Hippo kinases MST1/2, partially rescued the defects caused by SOX9 loss, linking SOX9 function experimentally to Hippo pathway state.

Phosphorylation-level Western blotting sharpened the mechanistic picture. SOX9 overexpression raised the ratio of phosphorylated to total YAP and lowered the phosphorylated-to-total MOB1 ratio, while SOX9 knockout produced the reciprocal pattern. XMU-MP-1 shifted both readouts toward the SOX9-overexpression signature, and total MOB1 remained unchanged across groups, indicating that the pathway rewiring was phosphorylation-dependent rather than a simple change in protein abundance. YAP and TAZ mRNA and protein levels rose with SOX9 gain and fell with SOX9 loss, and drug treatment partially restored them in SOX9-deficient cells, extending the transcriptomic associations to protein-level pathway readouts.

In vivo, the story held. Subcutaneous xenografts in nude mice showed that SOX9 overexpression significantly accelerated U87-derived tumor growth from day 14 onward, while SOX9 knockout markedly suppressed U251-derived tumors. XMU-MP-1 treatment further enlarged SOX9-overexpressing tumors and partially reversed the growth inhibition caused by SOX9 loss. Histopathology mirrored these dynamics: SOX9-overexpressing tumors displayed increased necrosis and nuclear atypia, whereas knockout tumors showed milder pathology, and the drug partially reversed both patterns. Ki67 immunohistochemistry confirmed the corresponding changes in proliferative activity, and CD68 staining revealed that myeloid and macrophage-like cell accumulation also shifted with SOX9 status, adding an immune dimension to the tumor microenvironmental effects.

Taken together, the study proposes what the authors call an early priming–late decoupling model. SOX9 acts early, at the moment of astrocyte-to-malignant conversion, to initiate Hippo–YAP/TAZ-linked malignant reprogramming. Once downstream transcriptional networks consolidate, the tumor becomes progressively less dependent on sustained SOX9 expression, which explains why stemness programs can persist in advanced disease even as SOX9 levels fall. The translational implication is pointed: therapies aimed at SOX9 may work best before malignant programs fully consolidate, whereas in later-stage tumors, blocking SOX9 alone may prove insufficient. The results argue for stage-specific and niche-aware strategies targeting the SOX9/Hippo/YAP–TAZ axis, particularly in the perivascular compartment where the axis is most strongly engaged. Limitations remain, including the reliance on public cohorts of limited size, established cell lines, and subcutaneous rather than orthotopic models, and the absence of YAP/TAZ nuclear localization data or a second pathway inhibitor. Even so, the study repositions SOX9 from a static stemness marker to a dynamic state-switch regulator, and it provides a conceptual framework for timing future interventions against one of medicine’s most stubborn cancers.

Subject of Research: The temporal role of SOX9 in priming Hippo–YAP/TAZ signaling during glioblastoma stemness acquisition

Article Title: Early SOX9 Activation Primes Hippo–YAP/TAZ Rewiring During Glioblastoma Stemness Acquisition

Article References: Mijiti, M., Li, Y., Maimaiti, A., Zhang, G., Lian, M., Song, C., Zhang, Y., Wang, J., Wang, Y., Chen, X., Kong, X., Xiaokaiti, X., Wusiman, N., Zhu, G., Geng, D., & Shi, H. (2026). Early SOX9 Activation Primes Hippo– YAP / TAZ Rewiring During Glioblastoma Stemness Acquisition. Journal of Cellular and Molecular Medicine, 30(17), Article e71341. https://doi.org/10.1111/jcmm.71341

Image Credits: AI Generated

DOI: 10.1111/jcmm.71341

Keywords: glioblastoma, SOX9, Hippo pathway, YAP/TAZ, glioma stem cells, pseudotime analysis, spatial transcriptomics, perivascular niche, XMU-MP-1, CRISPR, tumor plasticity, xenograft