INTRODUCTION:The ability to distinguish between transcripts that differ by a single nucleotide positions our padlock assay as a highly accurate imaging tool for detecting disorders associated with structural variations in the human genome. In this study, we evaluated the effectiveness of the padlock assay in identifying TTR gene variants in a case of transthyretin amyloidosis (ATTR), a rare multisystemic disease. ATTR may result from autosomal dominant mutations in the TTR gene or occur in a wild-type form. METHODS:We applied the padlock assay in combination with rolling circle amplification (RCA) and fluorescence microscopy, using peripheral blood mononuclear cells (PBMCs) as clinical samples. RESULTS:Using the padlock assay, 1) we detected intracellular TTR transcripts in 80% of PBMCs, including a benign variant caused by a single nucleotide substitution in intron 3, 2) we visualized the subcellular localization of both coding and non-coding regions of TTR transcripts, and 3) through dual staining, we simultaneously detected both wild-type and mutated TTR intron 3 in patient- derived cells. Additionally, we found that PBMCs and platelets are immunoreactive to TTR antibodies, suggesting that immunocompetent cells may contribute to the distribution of TTR protein across tissues and organs. DISCUSSION:We demonstrate that the padlock assay can serve as a non-invasive imaging test capable of spatially detecting genomic variants in ATTR. CONCLUSION:These findings suggest that the padlock assay has potential application in evaluating the efficacy of disease-modifying therapies in extra-hepatic cells. Moreover, this study is the first to highlight PBMCs as a valuable source for advancing our understanding of ATTR pathogenesis and for supporting the development of improved therapeutic approaches.

Tumor-induced osteomalacia (TIO) is an ultrarare paraneoplastic syndrome of abnormal phosphate and vitamin D metabolism secondary to the overproduction of fibroblast growth factor 23 by small-sized mesenchymal tumors typically located in soft tissues and bone. The tumor has adverse effects on bone and patients complain of skeletal symptoms and, in severe cases, they suffer multiple devastating fractures. Specific features may characterize the histology of tumors located in bone with respect to those found in extra-skeletal sites. Indeed, the matrix may contain foci resembling primitive cartilage and osteoid. Light microscopy of bone biopsy samples reveal accumulation of osteoid due to thickening of osteoid seams and, if tetracyclines were sequentially administrated, fluorescence microscopy reveals prolongation of the mineralization lag time. Areal BMD assessed by DXA is significantly lower at both the lumbar and femoral sites in patients with TIO and values of trabecular bone score are significantly reduced with respect to healthy individuals. Patients with TIO are also characterized by significant impairment in bone quality at both the trabecular and cortical compartment when evaluated by HR-pQCT. Successful surgical removal of the causative tumor completely reverts biochemical abnormalities. BMD accrual is impressive in the short term at the central (spine and hip) level but may take longer to improve, together with microstructural parameters, at peripheral sites (radius and tibia). Future studies should address effects of long-term treatment on quality-of-life outcomes related to irreversible events, such as vertebral fractures. This is particularly important in patients with a heavy burden due to a long-standing disease.

Elevated expression of THBS1 and THBS2 in intrahepatic cholangiocarcinoma (iCCA) contributes to tumor growth and metastatic dissemination. Both proteins are predominantly produced by cancer-associated fibroblasts (CAFs) and iCCA cells, enhancing the interaction of malignant cholangiocytes with the extracellular matrix (ECM). Here, we identify integrin α3β1 and α6β1 as the cognate receptors for THBS1 and THBS2 on iCCA cell surface. Disruption of the THBS1-integrin β1 axis via monoclonal antibodies, THBS1-derived peptides, or THBS1 knockout (KO) iCCA cells reduces autocrine and paracrine integrin β1 activation, resulting in decreased ECM adhesion in both two-dimensional and three-dimensional assays. Loss of endogenous THBS1 also alters cell morphology, weakens intracellular junctions, and prevents tumor formation in mouse xenograft models. These findings identify the THBS1/THBS2-integrin β1 axis as a key driver of iCCA cell adhesion and malignancy, supporting its potential as a therapeutic target for iCCA-specific interventions.

Abstract Bone marrow stromal cells (BMSC) – which include skeletal stem cells – are a promising tool in regenerative medicine. However, their heterogeneous and unpredictable in vivo behaviour remains a critical barrier preventing the development of standardized therapeutic approaches for skeletal tissue regeneration. Several studies have attempted to identify in vitro features that could correlate with the in vivo differentiation properties, yet the mechanisms ruling BMSC heterogeneity remain poorly understood. Here, using time-lapse imaging, we lineage-trace 32 single-cell-derived BMSC colonies through seven generations. We observe significant inter-colony and intra-colony heterogeneity in lineage topology (determined by the number of senescent or apoptotic cells) and in replicative kinetics (measured from proliferating cells only). Interestingly, topology and kinetics are strongly correlated, suggesting the existence of regulatory factors linking the non-dividing/apoptotic subpopulations with proliferating cells. Furthermore, BMSCs display a high degree of cell-cycle synchronization during early generations, indicating stage-specific regulatory mechanisms through which cells influence each other. By employing a non-interacting population growth model, we demonstrate that the observed synchronization cannot be explained by an uncorrelated branching process; instead, correlated division times among cells must be present. Our findings reveal fundamental mechanisms governing BMSC heterogeneity and growth dynamics that may inform strategies to control their regenerative potential.

BACKGROUND Temporal bone necrosis is a rare condition, most commonly caused by radiation therapy, bisphosphonate treatment, and osteomyelitis. Cholesteatoma can contribute to temporal bone necrosis, particularly in the presence of secondary ear infections. We report a case of extensive temporal bone necrosis with intracranial involvement in a patient with mastoid and petrous apex cholesteatoma, further complicated by actinomycosis. CASE REPORT A 47-year-old woman presented with persistent otorrhea and right-sided conductive hearing loss. Imaging demonstrated diffuse necrotic tissue extending from the retroauricular soft tissues to the middle ear and the petrous apex of the temporal bone, with erosion of the tegmen tympani. An extended petrosectomy was performed with particular attention to preserving the facial nerve. Histopathologic examination revealed the coexistence of cholesteatoma, necrotic bone sequestra, and colonies of Actinomyces. Postoperatively, the patient completed a course of intravenous antibiotics and adjuvant hyperbaric oxygen therapy. At 1-year follow-up, no evidence of recurrence was observed on radiological and clinical evaluation. CONCLUSIONS This case underscores an unusual presentation of temporal bone necrosis, occurring in the absence of conventional predisposing factors. The concomitant presence of cholesteatoma and Actinomyces infection highlights the importance of considering atypical etiologies in progressive otologic disease. Early diagnosis, comprehensive surgical debridement, and multidisciplinary management were essential to achieving favorable clinical and radiological outcomes.

Tumor-induced osteomalacia (TIO) is an ultrarare paraneoplastic syndrome caused by overproduction of fibroblast growth factor 23 (FGF23). There is low awareness of the disease so that TIO is underdiagnosed. In addition, symptoms reported by patients with TIO are usually nonspecific thus rendering the diagnosis difficult to be made. Biochemical hallmarks are represented by hypophosphatemia, increased or inappropriately normal levels of FGF23, low to low normal circulating 1,25(OH)2D and renal phosphate wasting. From the histological point of view, phosphaturic mesenchymal tumors are the entities responsible in majority of cases. Tumors causing TIO are generally of small size and grow slowly. There are a number of functional and anatomical imaging techniques utilized for tumor localization; 68Ga DOTA based technologies have the better sensitivity and specificity in respect to other techniques. Surgery is the treatment of choice; in a very few selected cases non pharmacological and pharmacological treatment are available.

Bone disorders and skeletal defects represent a significant clinical challenge, often requiring transplantation techniques limited by donor site morbidity and insufficient regenerative potential. Tissue engineering and regenerative medicine (TERM) strategies using 3D bioprinting have emerged as promising alternatives, but their efficacy is limited by the difficulty of directing stem cell differentiation in a controlled and reproducible manner. To address this limitation, we are proposing 3D bone printing via ultrasound-mediated osteogenic differentiation of stem cells (referred to here as '3DBonUS'). This biofabrication approach integrates low-intensity pulsed ultrasound (LIPUS) with a microfluidic-assisted 3D bioprinting system. This unprecedented approach enables biophysical stimulation of human bone marrow stromal cells during the fabrication of scaffolds, promoting osteogenic differentiation without the need for extensive post-fabrication treatments. In addition, the incorporation of microbubbles enhanced the effects of LIPUS by amplifying mechanical signals at the cellular level. Our results revealed that the 3DBonUS system significantly upregulated key osteogenic markers (RUNX-2, alkaline phosphatase (ALP), COL1A1, bone morphogenetic protein-2, OCN and OPN) as confirmed by immunofluorescence and reverse transcription polymerase chain reaction analysis. Moreover, the LIPUS-treated constructs showed a significant (p< 0.05) increase in ALP activity and calcium deposition, indicating enhanced mineralisation. The biofabricated constructs maintained high cell viability while exhibiting improved osteogenic differentiation, surpassing traditional 3D bioprinting approaches in both efficiency and efficacy. The 3DBonUS strategy represents a new modality in skeletal TERM, combining biofabrication with targeted mechanical stimulation, with potential for scalability of scaffold manufacturing and clinical application. Future studies will aim to validate the functional skeletal scaffoldsin vivoto assess their regenerative potential, with the goal of advancing patient-specific bone implants with enhanced osteogenic properties.

Bone marrow stromal cells (BMSC) – which include skeletal stem cells – are a promising tool in regenerative medicine. However, their heterogeneous and unpredictable in vivo behaviour remains a critical barrier preventing the development of standardized therapeutic approaches for skeletal tissue regeneration. Several studies have attempted to identify in vitro features that could correlate with the in vivo differentiation properties, yet the mechanisms ruling BMSC heterogeneity remain poorly understood. Here, using time-lapse imaging, we lineage-trace 32 single-cell-derived BMSC colonies through seven generations. We observe significant inter-colony and intra-colony heterogeneity in lineage topology (determined by the number of senescent or apoptotic cells) and in replicative kinetics (measured from proliferating cells only). Interestingly, topology and kinetics result strongly correlated, suggesting the existence of regulatory factors linking the non-dividing/apoptotic subpopulations with proliferating cells. Furthermore, BMSCs display highly synchronized cell cycles during early generations, indicating stage-specific regulatory mechanisms through which cells influence each other. By employing a non-interacting population growth model, we demonstrate that the observed synchronisation cannot be explained by an uncorrelated branching process; instead, cell-to-cell correlation of division times must exist. Our findings reveal fundamental mechanisms governing BMSC heterogeneity and growth dynamics that may inform strategies to control their regenerative potential.

Pigment epithelium-derived factor (PEDF) is a multifunctional soluble glycoprotein, primarily known for its potent anti-angiogenic properties. In recent years, its ability to counteract cell proliferation and motility has generated interest in PEDF as a potential tumor suppressor. In the intrahepatic Cholangiocarcinoma (iCCA), PEDF, Thrombospondin 1 (THBS1), and Thrombospondin 2 (THBS2) are expressed and released into the tumor microenvironment (TME), where they promote lymphangiogenesis at the expense of the neoangiogenic program, aiding the dissemination of cancer cells via lymphatic vessels. Recently, we demonstrated that THBS1 and THBS2 directly affect iCCA cells, exacerbating their malignant behavior, while the direct role of PEDF remains to be elucidated. In this study, through a cell-based assay and molecular analysis, we investigate the direct function of PEDF on two well-established iCCA cell lines. Our results show that PEDF affects cancer cell motility in a paracrine manner, reducing their migratory and invasive capabilities. Notably, our data suggest that the PEDF-induced inhibition of motility in iCCA cells occurs through the MAPK/ERK signaling pathway, as indicated by the reduced phosphorylation of ERK1/2. Overall, this study provides the first evidence of PEDF acting as a tumor suppressor in iCCA.

Management of patients with head and neck cancer (HNC) is a complex process that involves extensive knowledge on (at least) the discipline of surgery, radiation oncology, medical oncology and molecular biology. It includes prevention, cancer treatment, follow up schedule and potential risk from treatment (radiation exposure). A major point is that perhaps now more than ever process management needs to be at the center of the HNC care. Process management transcends all specialties: it should be recognized as a component of the team to embrace rather than fear change. Process management can guarantee the quality of the decisional process and the technical quality of the equipment, such as access to care for all, implementation of technology to control data entry, standardization of procedures and safety. Each step of the process needs adequate controls to avoid errors, ensure optimal treatment strategy and increase patient satisfaction. The application of process management method can lead to reduce treatment start times, fall risk and therefore, improve quality of HNC patient care and safety.

Mucopolysaccharidosis type I (MPS-I) is a rare pediatric disease caused by mutations in the α-L-iduronidase (IDUA) gene encoding for a lysosomal enzyme involved in glycosaminoglycan metabolism. While newborns with the severe Hurler variant are usually asymptomatic at birth, progressive disease manifestations emerge early in life. Since previous studies on lentiviral vector gene therapy (GT) in Hurler patients have demonstrated superior metabolic correction and early beneficial clinical effects, we investigated whether applying this GT approach during the neonatal period could be effective in preventing disease pathology before it becomes irreversible. Thus, newborn MPS-I mice were transplanted with affected bone marrow-derived progenitor cells transduced with an IDUA-encoding lentiviral vector. Treated animals displayed increased IDUA levels, significantly reducing substrate accumulation in analyzed organs, indicating metabolic correction. Skeletal manifestations, typically resistant to conventional therapies, showed improvements at radiographic and histological levels post-treatment. Additionally, a decrease in brain cortex vacuolization and inflammation suggested neurological amelioration. Overall, this study provides a proof of principle demonstrating the effectiveness of neonatal ex vivo GT in MPS-I mice and supports its potential for further optimization at the pre-clinical level.

Human bone marrow stromal cells (BMSC) include skeletal stem cells with ground-breaking therapeutic potential. However, BMSC colonies have very heterogeneous in vivo behaviour, due to their different potency; this unpredictability is the greatest hurdle to the development of skeletal regeneration therapies. Colony-level heterogeneity urges a fundamental question: how is it possible that one colony as a collective unit behaves differently from another one? If cell-to-cell variability were just an uncorrelated random process, a million cells in a transplant-bound colony would be enough to yield statistical homogeneity, hence washing out any colony-level traits. A possible answer is that the differences between two originating cells are transmitted to their progenies and collectively persist through an hereditary mechanism. But non-genetic inheritance remains an elusive notion, both at the experimental and at the theoretical level. Here, we prove that heterogeneity in the lineage topology of BMSC clonal colonies is determined by heritable traits that regulate cell-cycle exit. The cornerstone of this result is the definition of a novel entropy of the colony, which measures the hereditary ramifications in the distribution of inactive cells across different branches of the proliferation tree. We measure the entropy in 32 clonal colonies, obtained from single-cell lineage tracing experiments, and show that in the greatest majority of clones this entropy is decisively smaller than that of the corresponding non-hereditary lineage. This result indicates that hereditary epigenetic factors play a major role in determining cycle exit of bone marrow stromal cells.

Bone disorders and skeletal defects represent a significant clinical challenge. Tissue engineering and regenerative medicine (TERM) strategies using 3D bioprinting have emerged as promising alternatives, but still limited by the inability of directing stem cell differentiation in a controlled and reproducible manner. Advancing beyond this, we are proposing 3D bone printing via primed differentiation of stem cells with ultrasound (referred to here as ‘3DBonUS’). This approach synergistically integrates low-intensity pulsed ultrasound (LIPUS) with a microfluidic-assisted 3D bioprinting platform enabling the biophysical stimulation of human bone marrow stromal cells (HBMSCs) during extrusion, promoting osteogenic differentiation without the need for post-fabrication treatments. Moreover, the incorporation of microbubbles enhanced the effects of LIPUS by amplifying mechanical signals at the cellular level. 3DBonUS was found to significantly upregulate key osteogenic markers (RUNX-2, ALP, COL1A1, BMP-2, OCN, OPN) as confirmed by immunofluorescence and RT-qPCR analysis. Furthermore, the LIPUS-treated constructs showed a significant increase in alkaline phosphatase activity and calcium deposition, indicating enhanced mineralisation. The 3DBonUS strategy represents a new modality in skeletal biofabrication, harnessing targeted minimally-invasive mechanical stimulation, with potential for manufacturing scalability and clinical application. Future studies will aim to validate 3DBonUS in vivo to assess the ultimate regenerative potential with enhanced osteogenic properties. ### Competing Interest Statement The authors have declared no competing interest. MTF BIOLOGICS, OSTEOMIMIC European Research Council, 855923 European Innovation Council, 101098989 Next Generation EU PNRR, J33C22001130001

Bone pain is a major symptom of many skeletal disorders. Fibrous dysplasia (FD) is a genetic disease with mono or polyostotic skeletal phenotype due to the post-zygotic occurrence of the causative Gsα mutation. Bone pain in FD often associates with skeletal deformities and fractures or nerve impingement by the pathological tissue. However, even in the absence of complications, FD patients often complain of a chronic pain that does not correlate with their disease burden. Multiple hypotheses have been made to explain this pain. However, its pathogenetic mechanisms remain, as yet, largely unexplored. In this study, we first demonstrate that the FD mouse model EF1α-GsαR201C develops behavioral impairments and altered response to nociceptive stimuli that, as in FD patients, do not correlate with their skeletal disease burden, thus providing a reliable model to study bone pain in FD. Then, we show that in EF1α-GsαR201C mice, the overall pattern of skeletal innervation is preserved and that within FD lesions, sensory fibers are variably and focally distributed, mainly at perivascular sites. Finally, we provide the first analysis of a series of human FD bone biopsies showing that, within the lesional tissue, sensory nerve fibers are few despite the rich vascular network and appear to be well-organized. These data show that, albeit sensory nerve fibers are found within FD lesions, bone pain in humans and functional impairment in mice are not associated to pathological sensory nerve sprouting or formation of neuromas in the Gsα-mutated skeleton.

Immunotherapy has a crucial role in the treatment of recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, only a small percentage of patients achieve long-term benefit in terms of overall response and survival. It was shown that HNSCC has an immunosuppressive microenvironment due to high levels of regulatory T cells and immunosuppressive molecules, such as LAG3 and CD73. The aim of our study was to investigate if the expression of CD73 by neoplastic and immune cells could affect the efficacy of anti-PD-1 immunotherapy. We reviewed data from 50 patients with R/M HNSCC receiving first line immunotherapy with or without chemotherapy based on a combined positive score (CPS). CD73 expression by cancer and immune cells was evaluated on pre-treatment and the percentage of stained cells was recorded. We analysed the association between CD73 expression on neoplastic and immune cells and early progression (EP), defined as progression occurring within 3 months. In 88 % of patients the primary tumour site was in the oral cavity or larynx. All patients received pembrolizumab associated in 40 % of cases to chemotherapy. CD73 was positive in 82 % and 96 % of cases on neoplastic and immune cells, respectively. The median value of CD73 was 32 % for neoplastic cells and 10 % for the immune ones. We observed a significant association between the CD73 expression on neoplastic cells over the median value and EP disease. We didn't record a correlation between the expression of CD73 on immune cells and early progression. Our findings suggest that higher expression of CD73 on neoplastic cells could predict resistance to immunotherapy in patients with CPS positive R/M HNSCC. The addition of this biomarker to routine evaluation of CPS could help to select the patients primary resistant to antiPD-1 immunotherapy.