Head and neck cancer (HNC) is the sixth most common cancer worldwide and remains associated with high mortality. Current therapeutic approaches are often aggressive and lead to functional impairment and disfigurement, reducing quality of life. Minimally invasive and more localized treatment strategies are needed. Gold nanoparticles (AuNPs)-mediated photothermal therapy (PTT) has emerged as a promising approach due to the high photothermal conversion efficiency of AuNPs and their ability to generate localized heat upon near-infrared (NIR) irradiation. This study evaluates the potential of AuNP-mediated PTT for HNC as a proof-of-concept. For this, the physicochemical and optical properties of the AuNPs were characterized. They demonstrated long-term stability, for up to one year, and photothermal stability, maintaining performance throughout four irradiation cycles. The efficacy of AuNPs-mediated PTT was assessed in vivo using two murine models differing in tumor location (neck or flank). Following intratumoral administration of AuNPs and repeated laser irradiation cycles, a clear photothermal effect was observed, associated with increased tumor necrosis and a trend toward reduced tumor progression. Biodistribution studies revealed an accumulation of the AuNPs at the tumor. Importantly, safety assessments showed no significant alterations, indicating the absence of systemic toxicity following treatment. Overall, the developed AuNPs demonstrated long-term physicochemical stability, robust photothermal performance, strong intratumoral accumulation and a favorable safety profile. These findings support the potential of AuNPs-mediated PTT as a localized and minimally invasive therapeutic strategy for HNC. Further studies will include the assessment of the long-term therapeutic outcomes to advance this approach towards clinical translation.

Canine cutaneous mast cell tumours (MCTs) demonstrate variable clinical behaviour. Histological grading remains the primary prognostic approach, but some low-grade tumours behave aggressively, and interobserver variability persists. This underscores the need for complementary and objective biomarkers. Raman spectroscopy (RS) is a non-destructive optical technique based on the inelastic scattering of monochromatic light capable of detecting subtle biochemical changes in tissues. This exploratory study evaluates whether RS could differentiate low-grade from high-grade cutaneous MCTs based on characteristic vibrational signatures. Eighteen canine cutaneous mast cell tumours were graded according to the Kiupel two-tier system, comprising 10 low-grade and 8 high-grade cases. Formalin-fixed tumour samples were analysed, yielding a total of 68 Raman spectra. Spectra were acquired with a 785 nm laser, pre-processed using baseline correction, and normalized to the 1007 cm-1 phenylalanine band. Data normality was tested with the Shapiro-Wilk test, and group comparisons used the Mann-Whitney U test (α = 0.05). The 1007 cm-1 band showed no significant difference between Low- and High-Grade tumours (p = 0.068), validating its use for normalization. Conversely, the 860 cm-1 tyrosine-associated band was significantly higher in high-grade tumours (p = 0.033). Ratios normalized to the 1007 cm-1 peak consistently discriminated between grades, with significantly higher values in low-grade tumours across multiple spectral regions-particularly 1007/1299 and 1007/1301-1320 (all p < 0.05). Overall, RS identified distinct biochemical fingerprints between tumour grades in canine cutaneous MCTs, especially in spectral regions associated with amino acids, lipids, and nucleic acids. These findings support the potential of RS as a complementary tool for MCT characterization and encourage further validation in larger cohorts with clinical outcome data.

Cutaneous manifestations of feline leishmaniosis may occur at anatomical sites commonly affected by squamous cell carcinoma (SCC), creating potential diagnostic overlap. Although concurrent Leishmania infection and SCC have been described in individual cats, the presence of parasite DNA in feline SCC tissue has not been systematically investigated. This retrospective cross-sectional study evaluated 219 formalin-fixed, paraffin-embedded feline SCC samples submitted to a veterinary diagnostic laboratory in Portugal between 2020 and 2023. Samples were screened using a Leishmania-genus-specific nested PCR targeting SSU rDNA. PCR-positive cases were subsequently analyzed at four additional loci (cytB, g6pdh, hsp70, and ITS rDNA) and reassessed histopathologically. Leishmania spp. SSU rDNA was detected in 3/219 samples (1.4%; 95% CI: 0.5–3.9%), corresponding to SCC lesions on the pinna, nasal planum, and eyelid. Molecular characterization was possible in one case, involving an SCC of the nasal planum. Phylogenetic analysis showed that the hsp70 sequence clustered with reference sequences of L. infantum, whereas the cytB sequence grouped within the L. donovani complex without species-level resolution. In the same lesion, structures morphologically compatible with amastigotes were observed within macrophages in the tumor-associated inflammatory infiltrate. Overall, Leishmania DNA was detected in only a small proportion of feline SCCs, and the findings support coexistence rather than an association between infection and tumor development. These findings have direct diagnostic relevance in endemic areas, where histopathologic confirmation of SCC should not preclude investigation for concurrent Leishmania infection when clinical or epidemiologic suspicion persists.

Mast cell tumors (MCT) are among the most common neoplasia in dogs, representing up to 21 % of skin tumors. However, etiology and risk factors for its development remain unclear. This study aimed to reduce this knowledge gap by comprehensively analyzing 905 MCT cases diagnosed in Portugal between 2019 and 2021, using descriptive and inferential analyses. Most tumors affected the skin, with 69.9 % and 21.2 % classified as cutaneous and subcutaneous tumors, respectively. Only subcutaneous MCT exhibited female predisposition. Breed-specific analyses revealed male predominance in French Bulldogs and female predominance in Shar-Peis. Tumors in the extremities were the most prevalent (43.2 %, n = 183). Age-related characteristics varied by breed, with Pugs, Boxers, French Bulldogs, and Shar-Peis being diagnosed at younger ages. Logistic regression showed that age increased the likelihood of developing higher-grade cutaneous tumors (p < 0.01, OR=1.17, 95 % CI 1.02-1.21) and subcutaneous tumors with an infiltrative pattern (p = 0.02, OR=1.17, 95 % CI: 1.04 -1.33). The estimated annual incidence risk for MCT in dogs from Lisbon and Set & uacute;bal districts is 3.1 cases per 10,000 dogs, and 3.0 for males and 3.2 for females. Compared to mixed-breed dogs, Boxers, Shar-Peis, and Golden Retrievers had significantly higher relative risks (7.1, 6.3, and 5.9, respectively, p < 0.01). Sex-specific relative risks showed Boxers with the highest values among males (9.9, p < 0.01) and Shar-Peis among females (8.0, p < 0.01). This study provides insights into canine MCT, emphasizing the importance of age, sex, and breed, as well as the need for tailored veterinary care that considers these demographic characteristics to enhance prevention, early detection, and management.

Multicellular organisms rely on inter-organ communication networks to maintain vital parameters within a dynamic physiological range. Macrophages are central to this homeostatic control system, sensing and responding to deviations of those parameters to sustain organismal homeostasis. Here, we demonstrate that dysregulation of iron (Fe) metabolism, imposed by the deletion of ferritin H chain (FTH) in mouse parenchymal cells, is sensed by monocyte-derived macrophages. In response, monocyte-derived macrophages support tissue function, energy metabolism, and thermoregulation via a mechanism that sustains the mitochondria of parenchymal cells. Mechanistically, FTH supports a transcriptional program promoting mitochondrial biogenesis in macrophages, involving mitochondrial transcription factor A (TFAM). Moreover, FTH sustains macrophage viability and supports intercellular mitochondrial transfer from donor parenchymal cells. In conclusion, monocyte-derived macrophages cross-regulate iron and energy metabolism to support tissue function and organismal homeostasis.

Melanoma is the most aggressive type of skin cancer and recently approved drugs are often associated with resistance and significant adverse effects. Therefore, the design of more effective and safe options remains imperative. Photothermal therapy (PTT) using gold nanoparticles (AuNPs) presents a promising and innovative approach. In this work, the efficacy of combining a previously optimized formulation of AuNPs coated with a mixture of hyaluronic and oleic acids (HAOA-AuNPs) with near-infrared (NIR) laser irradiation in melanoma cell lines was explored. Coated and uncoated AuNPs formulations were characterized in physicochemical, morphological and elemental terms. Next, the cellular uptake efficiency as well as antiproliferative activity of the combination of each formulation with laser irradiation was evaluated. Subsequently, HAOA-AuNPs were selected to assess the underlying mechanism of combined therapy by cell cycle and Annexin V/PI assays. An in vivo syngeneic murine melanoma model was also conducted. In vitro studies demonstrated that 24 h after incubation and in the absence of laser, HAOA-AuNPs did not exhibit cytotoxic effects on the melanoma cell lines tested, similar to the laser alone. On the contrary, the combination therapy resulted in a large reduction in cell viability. Furthermore, it has been shown to promote S-phase cell cycle arrest and increase in the percentage of late apoptotic cells. Finally, the in vivo proof-of-concept showed that the intratumoral administration of HAOA-AuNPs followed by three laser irradiations impaired tumor progression. Collectively, AuNP-based PTT holds significant potential to improve treatment efficacy and safety, offering a versatile and potent tool against cancer.

Multicellular organisms rely on inter-organ communication networks to maintain vital parameters within a dynamic physiological range. Macrophages are central to this homeostatic control system, sensing deviations of those parameters and responding accordingly to support tissue function and organismal homeostasis. Here we demonstrate that dysregulation of iron metabolism in parenchyma cells, imposed by the deletion of ferritin H chain, is sensed by monocyte-derived macrophages. In response, macrophages derived from circulating monocytes support tissue function, energy metabolism and thermoregulation, as demonstrated in bone marrow chimeric and parabiotic mice. This salutary effect is contingent on a transcriptional program, controlled in macrophages by the transcription factor A mitochondria. This transcriptional response acts in a non-cell autonomous manner to support the mitochondria of parenchyma cells, irrespectively of mitochondrial transfer. In conclusion, monocyte-derived macrophages cross-regulate Fe and energy metabolism to support tissue function and organismal homeostasis. ### Competing Interest Statement The authors have declared no competing interest.

Classification schemes regarding canine subcutaneous mast cell tumors (csMCTs) remain elusive, lack consensus, and are prone to interobserver variability and bias. This observational study aimed to assess the reproducibility and the prognostic significance of volume-weighted mean nuclear volume ((v) over bar (v)), a stereological estimation offering insights into nuclear size and its variability, in csMCTs. Thirty csMCTs were selected with information regarding outcome, and (v) over bar (v) was estimated using the "point-sampled intercept" method. Interobserver and intraobserver (v) over bar (v) reproducibility yielded concordance coefficients near or above 0.90. Regarding previously reported risk factors (pattern, mitotic count, and multinucleated cells), no statistically significant differences were identified between patterns and clinical outcome, nor between patterns and (v) over bar (v); however, the infiltrative pattern was represented more in the poorer outcome group and had higher (v) over bar (v) values. When comparing (v) over bar (v) and clinical outcome, a statistically significant difference emerged. Cases with poorer outcomes had higher (v) over bar (v)values ((x) over tilde = 192.9) than cases with more favorable outcomes ((x) over tilde = 120.5), and this association was statistically significant on both univariable and multivariable analyses. This study suggests that (v) over bar (v) is highly reproducible and is associated with clinical outcome in csMCTs.

Pre-metastatic niche (PMN) formation is a critical step in metastatic progression. However, the biological effects of subtherapeutic doses of ionizing radiation (SDIRs) following radiotherapy on this process remain unclear. Using a 4T1 breast cancer mouse model, we investigated the effects of SDIRs (3 × 0.3 Gy) on lung PMN development and metastasis upon SDIR exposure on days 8–10 post-tumor injection, followed by mastectomy and analyzed on day 24. SDIRs significantly increased the total metastatic volume (TMV) in lungs, suggesting an accelerated PMN formation. Mechanistically, the SDIR acted as an early catalyst for niche priming, upregulating Bv8 expression, enhancing neutrophil recruitment, and increasing MMP9, S100A8, and Il6 production in the PMN by day 11. Moreover, SDIR drives metastasis through distinct mechanisms. Proteomic analysis revealed SDIR-driven metabolic reprogramming, with a shift away from fatty acid metabolism toward glycolysis and lipid accumulation within the PMN. This shift contributes to extracellular matrix (ECM) remodeling, immune modulation, and the upregulation of adhesion-related pathways, shaping a microenvironment that accelerates metastatic outgrowth. By reprogramming the pre-metastatic lung, the SDIR highlights the need to integrate organ-specific radiation exposure into metastasis models. Metabolic and immune-stromal pathways emerge as potential therapeutic targets, underscoring the importance of refining radiotherapy strategies to mitigate unintended pro-metastatic effects.

Gold nanoparticle (AuNP)-mediated photothermal therapy (PTT) has emerged as a promising approach for cancer treatment, offering high precision, minimal invasiveness, and selective tumor ablation through localized hyperthermia. This review explores the fundamental principles of AuNP-mediated PTT, including the design of AuNPs, synthesis strategies, and surface modifications that enhance their biocompatibility and targeting efficiency. Additionally, the integration of AuNPs with other therapeutic modalities is also discussed, such as chemotherapy, radiotherapy, and immunotherapy, to achieve synergistic treatment outcomes. The theragnostic potential of AuNPs, combining diagnostic imaging with therapeutic applications, is also highlighted, emphasizing their role in personalized medicine. But, despite significant advancements, challenges such as biodistribution, long-term safety, and regulatory approval remain key barriers to clinical translation. Ongoing research is expected to refine AuNP-based PTT, paving the way for its widespread clinical application in cancer treatment.

Cognitive judgement bias in decision-making under ambiguity occurs both in animals and humans, with some individuals interpreting ambiguous stimulus as positive (optimism) and others as negative (pessimism). We hypothesize that judgement bias is a personality trait and that individuals with a pessimistic bias would be more reactive to stressors and therefore more susceptible to stress-related diseases than optimistic ones. Here, we show that zebrafish judgment bias is a consistent behavioral trait over time, and that pessimistic and optimistic fish express phenotype-specific neurogenomic responses to stress. Furthermore, both phenotypes show differential activation of the hypothalamic-pituitary-interrenal axis in response to chronic stress, suggesting that optimists have a lower stress reactivity. Accordingly, optimists seem to be more resilient to disease than pessimists, as shown by a lower tumorigenesis in a zebrafish melanoma line [Tg(mtifa:HRAS-GFP)]. Together these results indicate that judgement bias is paralleled by differences in the stress response with implications for disease resilience.

Cell competition in the thymus is a critical tumor suppressor mechanism that prevents leukemia. Here, we show that suboptimal bone marrow correction of γc -deficient mice triggers thymus autonomy and subsequent T cell acute lymphoblastic leukemia (T-ALL). γc -deficiency results in severe combined immunodeficiency (SCID) in mice and humans, and correction is achieved by hematopoietic stem and progenitor cell (HSPC) transplantation. Following inefficient bone marrow correction of γc -deficient mice, thymus function was intermittent, consistent with sporadic thymic colonization events. Thymocyte composition and phenotype changed, with the appearance of an aberrant TCRβ-CD4+CD8+ population that preceded leukemia. Leukemia onset and incidence were worsened if HSPCs were pre-cultured to mimic conditions used for gene editing and ameliorated if the hosts could not support thymopoiesis immediately. Lastly, the level of bone marrow reconstitution was crucial to inhibit thymus autonomy. Altogether, our data reveal that inefficient bone marrow correction directly impairs cell competition in the thymus and enables leukemia, underscoring the need to consider and preempt thymus autonomy when treating immunodeficiency. ### Competing Interest Statement The authors have declared no competing interest. La Caixa Foundation, LCF/PR/HR22/52420023 Fundação para a Ciência e Tecnologia, https://ror.org/00snfqn58, PTDC/MED-IMU/3649/2021, CEECIND/03106/2018, UI/BD/154901/2023, PD/BD/114341/2016, PD/BD/139190/2018

This study aimed to determine the agreement between margin status in feline mammary tumors’ surgery by comparing 3 margin assessment techniques and to determine the predictive value of prognostic clinical and histological parameters for margin status using intraoperative margin assessment techniques. During the intraoperative period, 69 surgical margins from 12 female cats undergoing unilateral radical mastectomy were assessed by imprint cytology (IC) and scrape cytology (SC), both intraoperative cytological techniques. The excised mastectomy specimens were then evaluated postoperatively by histopathology (HP). Cochran’s Q test was used to determine differences in positive margin detection for the three methods. The agreement between the three methods was assessed using the Fleiss kappa coefficient. A binary logistic regression model was used to assess whether clinical and histological prognostic parameters could predict intraoperative margin status. There was no significant difference in positive margins between the three methods (p=0.174). The agreement between the three methods was poor (Fleiss kappa = −0.020; 95% CI −0.156 to 0.117). Lymphovascular invasion is a significant predictor of a positive intraoperative margin, with an OD of 18.652 (95% CI = 1.742–199.652). These findings suggest that IC and SC are viable methods for assessing the status of surgical margins during surgery for feline mammary tumors. Intraoperative cytology may be complementary to HP, particularly in cases of lymphovascular invasion.

Chronic wounds (CWs), particularly in diabetic patients, remain a major clinical challenge because of impaired angiogenesis and fibroblast dysfunction. This study investigated the therapeutic synergy between low-dose ionizing radiation (LDIR; 0.3 Gy) and prevascularized spheroids composed of human dermal fibroblasts (HDFs) and endothelial colony-forming cells (ECFCs) embedded in a xeno-free fibrin hydrogel. In vitro, LDIR enhanced endothelial sprouting and fibroblast outgrowth from HDF-ECFC spheroids without affecting morphology or viability. Gene and protein analyses revealed LDIR-induced upregulation of angiogenic and profibrotic markers, including VEGFR2, CD31, VE-cadherin, COL4A2, α-SMA, TGF-β1 and LH2. Conditioned media from irradiated spheroids exhibited a proangiogenic secretome and significantly increased neovascularization in the chorioallantoic membrane assay. Notably, spheroids composed exclusively of fibroblasts failed to exhibit these LDIR-induced effects, highlighting the importance of endothelial-stromal interactions. Importantly, in vivo, the combination of nonirradiated HDF-ECFC spheroids followed by local LDIR significantly accelerated wound healing and restored skin remodeling, skin architecture and histological integrity by reducing pathological skin remodeling in the db/db diabetic mouse model. In contrast, neither LDIR per se nor preirradiated spheroids on their own promoted wound closure or histological repair. These findings demonstrate that LDIR enhances the regenerative capacity of prevascularized dermal spheroids through molecular activation, paracrine signaling and stromal‒endothelial crosstalk but requires in vivo irradiation of the wound niche to achieve therapeutic benefit. This combinatorial strategy offers a clinically translatable approach to modulate the wound microenvironment and overcome current limitations in CW management.

Background/Objectives: Head and neck cancer (HNC) is the sixth most common cancer worldwide, with a high mortality, particularly from head and neck squamous cell carcinoma (HNSCC). Although some therapeutic strategies are available, they might cause severe side effects. For example, surgery may result in disfigurement and functional loss, severely impacting the patient’s quality of life. Thus, minimally invasive and more effective alternatives are needed. Gold nanoparticle (AuNP)-mediated photothermal therapy (PTT) is a promising approach for HNC, which relies on AuNP photothermal efficiency and tumor localization. This study aimed to synthesize and characterize AuNPs, evaluate their safety without laser activation, and assess their efficacy with laser activation. Methods and Results: Their physicochemical and photostability over three months and sterility were confirmed. In vitro safety was tested using human non-cancerous and HNC cell lines, while in vivo biocompatibility was evaluated in the hen’s egg chorioallantoic membrane (CAM) model, with no adverse effects observed. Upon laser activation, AuNPs reduced HNC cell viability by 50–70%, including HNSCC lines. In vivo biodistribution studies showed that AuNPs remained at the injection site for up to one month without toxicity. Conclusions: Overall, the developed AuNP formulation demonstrates stability, biocompatibility, and prolonged local retention, key attributes for effective and targeted PTT. These findings support the potential of AuNP-mediated photothermal therapy as a promising treatment modality for HNC, although further preclinical and clinical studies are needed to optimize treatment parameters.

Buccal mucosa graft (BMG) urethroplasty is the gold standard for urethral stricture repair; nevertheless, graft failure remains challenging because of inadequate vascularization, excessive fibrosis and a dysregulated immune response. Low-dose ionizing radiation (LDIR) and low-intensity shockwave therapy (LiSWT) modulate angiogenesis, inflammation and tissue remodeling. However, their impact on BMG healing remains unexplored. This study aimed to assess the effects of LDIR and LiSWT applied separately on BMG integration, with a focus on vascularization, fibrosis, immune modulation and epithelial remodeling. Wistar Han IGS rats underwent urethral stricture induction followed by BMG urethroplasty. Animals were assigned to separate treatment groups receiving either LDIR (0.3 Gy) administered on postoperative days 2–5 or LiSWT (500 impulses, EFD: 0.160 mJ/mm²) applied on days 0, 7 and 14. Graft area perfusion was measured via laser Doppler imaging over time. Histology and spatial transcriptomics were performed on day 21 to assess vascularization, fibrosis, epithelial remodeling, inflammation and molecular signatures. LDIR significantly increased graft perfusion (mean + 68

Gold nanoparticles (AuNPs)-mediated Photothermal Therapy (PTT) is a minimally-invasive therapeutic approach that uses AuNPs to convert light into heat, leading to the thermal ablation of tumors. Thus, the efficacy of this strategy strongly relies on the photothermal conversion potential of AuNPs. The ability to convert light into heat can be enhanced by tuning the physicochemical and optical properties of AuNPs. This can be achieved by changing the conditions of AuNP's synthesis, such as the order of addition of reagents. The present work entails to explore how varying the order of reagents addition modulates the properties of AuNPs, particularly enhancing the photothermal conversion potential of the resulting AuNPs and consequently, improving PTT efficacy. For this, eleven different AuNPs' nanoformulations were synthetized following different sequences of addition of reagents. These nanoformulations were characterized regarding their physicochemical properties namely size, surface charge, gold concentration, surface morphology and maximum absorbance wavelength. In addition, their thermal activation profiles were determined in vitro. . Furthermore, the biocompatibility of different nano- formulations was also assessed. Three nanoformulations, with the most favorable photothermal activation profiles (AuNPs 2, 3 and 7), were then selected for preliminary in vitro safety and efficacy assays using a panel of cell lines. These three nanoformulations were deemed safe in vitro at the tested concentrations. At 250 mu M of gold content, and after an incubation period of 4 h, followed by 5 min irradiation with a laser emitting at 808 nm (7.96 W/cm2), 2 ), AuNPs 7 significantly reduced the cell viability of all cancer cell lines tested (MCF-7, HCT-116 and A375) by >= 45 %. However, such cytotoxic effect was not observed for the human keratinocyte cell line (HaCat), thus demonstrating its specificity towards cancer cells. Overall, the results herein presented reinforce that the order of reagents addition is highly important for achieving adequate AuNPs for PTT.