Rab24 protein levels show dynamic changes in mouse tissues and human cancers

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G. Mauricio Ramm, Farhad Ahmed, Sadaf Fazeli, Martin Alexander Lopez, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6176716/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Cell and Tissue Research → Version 1 posted 10 You are reading this latest preprint version Abstract Rab24 is an unusual member of the Rab family of small GTPases, implicated in autophagy, endocytosis and cell division. In order to elucidate possible organ and age-specific roles of Rab24, we investigated tissue-specific levels of Rab24 in mice by western blotting and immuno­histo­chemistry in samples from postnatal day one to 9 months of age. In adult mice, the highest protein levels were found in the brain followed by the kidney, while Rab24 levels in the pancreas, spleen, liver, lung, heart, and skeletal muscle were lower. Dynamic changes in Rab24 levels were observed during early postnatal development, with a sharp increase in the brain at postnatal day 14, after which the level remained high into adulthood. In the heart, skeletal muscle, pancreas and liver, higher Rab24 levels were observed during the first two postnatal weeks, after which the levels dropped and stayed low until adulthood. The age-dependent changes suggest organ-specific roles for Rab24 in development and maturation. Immunohistochemistry of the brain revealed that Rab24 was mostly present in neuronal cells. Also, epithelial cells in several tissues showed high Rab24 levels. These results suggest roles for Rab24 in neuronal and epithelial maintenance. Furthermore, we also analysed immunohistochemical staining for RAB24 in human cancers and normal tissues. RAB24 staining in cancers of the breast and skin was higher than in the corresponding normal tissues, while it was reduced in cancers of the digestive system and the urinary tract. In pancreatic neuroendocrine tumours that originate from islet cells, RAB24 levels were lower than in normal pancreatic islet cells. Collectively, our findings provide a comprehensive overview of RAB24 levels across a wide spectrum of human cancers. The observed differences in RAB24 levels between cancer types and between malignant and normal tissues, suggest that RAB24 may play context-dependent roles in malignancy. Rab24 Mouse tissues Cancer Pancreatic neuroendocrine tumours Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Rab GTPases regulate intracellular membrane trafficking events ranging from vesicle formation, vesicle transport, membrane tethering and membrane fusion. Rab24 was first described in 1993 to localize to the endoplasmic reticulum (ER), Golgi apparatus and late endosomes (Olkkonen, et al., 1993 ). It is an unusual member of the Rab family due to the presence of an atypical amino acid in the GTP-binding region (Erdman, et al., 2000 ). We and others showed that Rab24 functions in autophagy (Munafo and Colombo, 2002 , Yla-Anttila, et al., 2015 ), a catabolic process that recycles organelles and aggregate-prone proteins by transporting them to lysosomes, thereby producing substrates for biosynthesis and energy production. The Q38P point mutation in Rab24, leading to degeneration of cerebellar Purkinje neurons, was identified as the cause of canine hereditary ataxia in Gordon setters and Old English sheepdogs (Agler, et al., 2014 ). The affected canine neurons accumulate autolysosomes and ubiquitin-protein aggregates, in agreement with our results on the role of Rab24 in autophagy (Yla-Anttila, et al., 2015 ). Further, Rab24 was shown to regulate endosomal degradation by interacting with the late endosomal protein Rab7 (Amaya, et al., 2016 ). Together, these results show that Rab24 is important for neuronal health, possibly by regulating the delivery of autophagic and endocytic cargo to lysosomes. In humans, RAB24 has been associated with fatty liver disease and hepatocellular carcinoma. Liver RAB24 levels positively correlate with body fat and are highly increased in the livers of obese patients with non-alcoholic fatty liver disease (NAFLD) (Seitz, et al., 2019 ). Rab24 knockdown in mouse liver decreased hepatic fat and reduced serum cholesterol levels in obese mice, confirming the link between Rab24 levels and liver fat accumulation. Furthermore, several studies show RAB24 to be overexpressed, and/or be associated with, poor prognosis in liver cancer (Chen, et al., 2017b , He, et al., 2002 , Yang, et al., 2020 , Zhang, et al., 2020 , Zhu, et al., 2020 ). RAB24 expression is increased in hepatocellular carcinoma (HCC) due to downregulation of miR-615-5p, which normally downregulates RAB24 expression (Chen, et al., 2017b ). Ectopic overexpression of RAB24 facilitated HCC cell motility, invasion and adhesion, accelerated cell cycle progression, reduced apoptosis, and facilitated epithelial to mesenchymal transition, while knockdown of RAB24 had opposite effects in all these assays (Chen, et al., 2017b ). These data show that RAB24 plays a significant role in promoting the malignant phenotype of HCC cells. Furthermore, high RAB24 expression is an unfavourable prognostic marker in prostate cancer (Hu, et al., 2020 ). On the contrary, RAB24 was reported to be an independent low-risk factor in pancreatic adenocarcinoma (Deng, et al., 2022 ). According to the Human Protein Atlas (proteinatlas.org, Uhlen, et al., 2015 ), RAB24 is classified as a potentially favourable marker in clear cell renal cell carcinoma and an unfavourable marker in glioblastoma. These findings highlight RAB24’s context-dependent prognostic value in different cancer types. Despite these advances, the expression patterns of Rab24 in different tissues and developmental stages have not been analysed. Knowledge on these patterns can provide insights into Rab24’s possible physiological roles and potential contributions to age-related diseases. Given that mice are commonly used as model organisms in autophagy research, and that Rab24 plays a role in autophagy, the understanding of the normal age-related expression patterns of Rab24 protein is important, as such patterns may serve as potential confounding factors when interpreting experimental results. In this study, we analysed Rab24 protein levels using western blotting in the brain, heart, liver, lung, kidney, spleen, skeletal muscle, and pancreas of C57BL/6 mice in different age groups from postnatal days to 9 months. Immunohistochemistry was also performed in order to analyse which cell types in these tissues express Rab24 protein. Our results revealed distinct cell and tissue-specific patterns and dynamic, age-dependent changes in Rab24 levels. This is the first study to provide a comprehensive record of Rab24 protein levels in mouse tissues of different age. In addition, we analysed tissue microarrays of different human cancers, where RAB24 levels differ from the normal tissue. Our findings provide a basis for further studies regarding the role of RAB24 as a prognostic or predictive factor. Material and methods Ethical compliance All experimental procedures involving animals were ethically reviewed and approved by the Project Authorization Board of the Regional State Administrative Agency of Southern Finland (ESAVE/613/2019), and complied with the guidelines of the Directive 2010/63/EU of the European Union. Sections from human cancer tissue microarrays (TMAs) were obtained from Helsinki Biobank, after the acceptance of the Ethics Committee of the Hospital District of Helsinki and Uusimaa (HUS/697/2020). The samples in the Helsinki Biobank are stored after receiving informed consent from all patients. The study conforms to the standards of the Declaration of Helsinki. Preparation of mouse tissue extracts and immunoblotting Mice were group housed under controlled temperature and 12-h light-dark rhythm with same-sex littermates, and given free access to food and water. C57BL/6 mice of different age (1 day, 7 days, 14 days, 1 month, 3 months, 6 months, and 9 months) were sacrificed by cervical dislocation (1, 7 and 14-day-old animals) or by carbon dioxide (1 month and older animals). Four mice, two males and two females, were used for each age group. Samples from the cerebral cortex, heart, liver, lung, kidney, spleen, skeletal muscle ( tibialis anterior ) and pancreas were collected and snap-frozen in liquid nitrogen. For protein extraction, approximately 35 mg of tissue was homogenized in 140 µl of homogenization buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1% NP-40 and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (A32959, Thermo Scientific). After adding a 5-mm stainless steel bead (69989, Qiagen), the samples were lysed using a TissueLyser LT (85600, Qiagen) at 50 Hz for 3 min. Subsequently, an additional 140 µl of homogenization buffer was added, bringing the total buffer volume to 280 µl per 35 mg of tissue. The lysates were rotated end-over-end at + 4°C for 1 h and centrifuged at at + 4°C, 16,000 g for 15 min. Protein concentration of the supernatants was determined by bicinchoninic acid (BCA) assay (23228, Thermo Scientific), and SDS sample buffer (100 mM sodium phosphate, pH 7.5, 2% w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol, 0.004% w/v bromophenol blue) was then added. The samples were heated at + 95°C for 4 min, and stored at -20°C. Per lane, 10 µg of total protein were resolved on a 12% SDS-PAGE gel and blotted to a polyvinylidene difluoride (PVDF) membrane (88518, Thermo Scientific). Total proteins were stained using TotalStain Q (AC2225, Azure Biosystems) and the blots were imaged using Azure Sapphire imaging system (Azure Biosystems). For antibody staining, the membranes were blocked with 5% non-fat milk powder in Tris-buffered saline (100 mM Tris-HCl, pH 7.6, 1.5 M NaCl) containing 0.05% Tween-20 (TBS-T). Membranes were probed with affinity-purified rabbit anti-RAB24 (11445-1-AP, Proteintech) in blocking solution at + 4°C overnight. Membranes were washed, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (111-035-003, Jackson ImmunoResearch) at room temperature for 1 h, and the bands were visualized with Clarity™ Western ECL Substrate (1705061, Bio-Rad). Blots were imaged using Azure Sapphire imaging system (Azure Biosystems), and the bands were quantified using Fiji/ImageJ (Schindelin, et al., 2012 ). In order to compare Rab24 levels between different blots, tissue extract from one 1-month-old liver was used as a control sample in the blots. The Rab24 signals were first normalized to total protein, and then normalized to the control sample in each gel. Preparation of mouse tissues for immunohistochemistry Immunohistochemistry was performed for mice aged 7 days and 1, 3 and 6 months. Cervical dislocation followed by decapitation was used for euthanisation of 7-day-old C57BL/6 mice. Older mice (1, 3 and 6 months) were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg), and transcardially perfused with phosphate-buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde (PFA) in PBS, using a peristaltic pump at a flowrate of 5 ml per minute. For all age groups, samples from the brain, heart, liver, lung, kidney, spleen, skeletal muscle and pancreas were collected and post-fixed in 4% PFA in PBS at + 4°C for 24 h. The samples were dehydrated, embedded in paraffin, and 5-µm sections were cut and mounted on glass slides. Immunohistochemical staining For Rab24 staining, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidases were quenched in 3% H 2 O 2 in methanol for 20 min. Antigen retrieval was performed in 10 mM citrate buffer, pH 6.0, by microwave heating at 640 W for 7 min and at 480 W for 7 min. Sections were permeabilized in 0.1% Triton X-100 in TBS-T for 5 min. Blocking was performed with 5% normal goat serum in TBS at room temperature for 1 h. Sections were incubated in affinity-purified rabbit anti-RAB24 (11445-1-AP, Proteintech) in 5% normal goat serum at + 4°C overnight. Subsequently, the sections were washed and incubated in biotinylated goat anti-rabbit IgG (PK-6101, Vector Laboratories) at room temperature for 1 h. Sections were washed, incubated with an avidin horseradish peroxidase complex (PK-6101, Vector Laboratories) in PBS at room temperature for 40 min, washed again and incubated in 3,3’-diaminobenzidine (DAB, ready-made reagent, SK-4100, Vector Laboratories) for 1 min. Slides were counterstained with Mayer’s haematoxylin (105.3 mM aluminium potassium sulphate, 3.308 mM haematoxylin, 505.3 µM sodium iodate, 4.758 mM citric acid, 302.2 mM chloral hydrate) for 1 min, dehydrated in a graded alcohol series and mounted using Pertex® mounting medium (00811, HistoLab). To confirm the specificity of the immunostaining, serial sections of the tissues were stained both with the protocol described above, and with a control protocol in which the primary antibody incubation was replaced by a prolonged blocking step (Fig. S1 ). The same control staining was done for the pancreatic neuroendocrine tumour sections (Fig. S2). For Ki-67 immunohistochemical staining, TMA sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed in 10 mM citrate buffer, pH 6.0, at + 99 o C for 20 min in PT Module (Lab Vision ™ ). Slides were set in Shandon ™ Coverplate system. Endogenous peroxidases were quenched in 3% H 2 O 2 in PBS for 10 min, and blocking was performed with 20% normal goat serum in PBS at room temperature for 20 min. Sections were incubated in rabbit monoclonal anti-Ki-67 (# RM-9106-S1, Thermo Scientific) in 1% BSA in PBS at room temperature for 60 min. Subsequently, the sections were washed and incubated in biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories) in PBS at room temperature for 30 min. Sections were washed, incubated with an avidin-HRP complex (PK-6100, Vector Laboratories) in PBS at room temperature for 30 min, washed again and incubated in DAB (BS04, ImmunoLogic a WellMed Company) for 6 min. Slides were counterstained with Harris haematoxylin (1.09253, Sigma-Aldrich), dehydrated in a graded alcohol series and mounted using Pertex® mounting medium. Control slide was incubated without the primary antibody. Images of whole slides were acquired using a Pannoramic 250 Flash slide scanner equipped with a 20x objective (3DHistech). Images were analysed and cropped using CaseViewer (3DHistech) and Fiji/ImageJ software (Schindelin, et al., 2012 ). Analysis of human tissue microarrays Paraffin sections from tissues microarrays (TMAs) containing samples from 75 different types of human cancers belonging to 220 patients, and from TMAs containing human pancreatic neuroendocrine tumour (PNET) samples from 120 patients were obtained from Helsinki Biobank. Part of the tissue cores also contained normal tissues that had been removed together with the tumours. The sections were stained immunohistochemically for RAB24 and Ki-67 as described above. Quantification of RAB24 staining intensity and Ki-67 labelling index was performed using QuPath software version 5.0.1 (Bankhead, et al., 2017 ). Whole-slide images were opened in QuPath and set to the H-DAB image type. A TMA grid was defined using the TMA dearrayer function. For the analysis of RAB24 staining intensity, RAB24-positive cells were detected and classified using the positive cell detection function. Tissue folds and other artifacts were manually excluded. In PNET samples, tumour tissue, pancreatic islets, and connective tissue were manually annotated in 15–20 representative areas, and the train object classifier function was used to classify the remaining tissues across the entire TMA. QuPath categorized the RAB24 staining intensity on a scale from 0 to 3, where 0 indicated no staining, 1 + weak staining, 2 + moderate staining, and 3 + strong staining. Each category had a separate threshold for the RAB24 staining intensity; the values for the threshold were selected manually using scores given by a pathologist as reference. RAB24 staining intensity was then quantified using the H-score, calculated as: H-score = 0 × (% negative cells, 0) + 1 × (% weakly positive cells, 1+) + 2 × (% moderately positive cells, 2+) + 3 × (% highly positive cells, 3+). The H-score data were exported using the show TMA measurement function. For the determination of Ki-67 index, whole-slide TMA images were processed as above in QuPath. A TMA grid was defined, and Ki-67-positive nuclei were detected using the positive cell detection function. Nuclei located in tissue folds were manually excluded. Tumour tissue and connective tissue were manually annotated in 10–20 representative areas in randomly selected cores, and an object classifier was trained to classify tissues throughout the TMA. The percentage of Ki-67 positive nuclei in tumour tissue was exported using the show TMA measurement function. Statistics Statistical analysis was performed with GraphPad Prism (GraphPad) using the tests indicated in the respective figure legends. Results Age-specific Rab24 levels in mouse tissues Rab24 is involved in autophagy, endocytosis, cell division and other essential cellular processes (Amaya, et al., 2016 , Militello, et al., 2013 , Yla-Anttila, et al., 2015 ), making its developmental regulation of particular interest. To compare Rab24 levels between organs in different age groups, we analysed samples from the brain (cerebral cortex), heart, liver, lung, kidney, spleen, skeletal muscle ( tibialis anterior ) and pancreas by immunoblotting. The tissues were obtained from mice spanning from early postnatal age to middle-aged adulthood (1-day to 9-month-old, Fig. 1 a). We utilized a total protein stain as a loading control for the quantifications. Total protein staining has been shown to be reliable for normalizing the loading in tissues with varied proteomic profiles, as it reflects the overall protein content consistently and compensates for tissue-specific differences (Bettencourt, et al., 2020 , Musyaju, et al., 2023 ). Firstly, we focused on comparing Rab24 levels across multiple tissues within each age group, providing insight into organ-specific Rab24 levels. For comparative analysis between the different blots, the relative levels of Rab24 in each blot were normalized to a control sample, obtained from the liver of a 1-month-old mouse, which was loaded onto each gel (Fig. 1 b-h). The liver was selected as the reference tissue due to its large size, which facilitates the preparation of ample, consistent cell extract. We did not observe any significant differences in Rab24 levels based on gender (data not shown). In 1-day and 7-day-old mice, Rab24 level varied between tissues, ranging from one fourth to 2-fold of the level in the liver control sample. The highest level was observed in the heart in both age groups (Fig. 1 b, c). A relatively high level was also observed in the brain at 1 day and 7 days, and in the kidney and skeletal muscle at 7 days. In contrast, the lowest Rab24 levels were detected in the spleen and pancreas at 1 day and 7 days, and the liver and lung at 7 days (Fig. 1 b, c). In 14-day and 1-month-old mice, Rab24 levels increased, with particularly elevated levels observed in the brain followed by the kidney, heart and skeletal muscle at 14 days, and the kidney at 1 month (Fig. 1 d, e). Conversely, the lung, spleen and pancreas showed low Rab24 levels. In 3-month-old mice, Rab24 levels of the brain showed a significant increase compared with the other tissues, which was consistent in the 6 and 9-month samples (Fig. 1 f-h). In addition, Rab24 levels remained elevated in the kidney in the 3, 6 and 9-month samples (Fig. 1 f-h). Overall, these findings revealed dynamic changes in Rab24 levels across different tissues during postnatal development and aging, suggesting potential tissue-specific functions for Rab24. Tissue-specific Rab24 levels according to age To enable more reliable comparisons of Rab24 levels within each organ during postnatal development and aging, we conducted an additional set of western blots in which all samples from each tissue were loaded on the same gel. This allowed us to minimize gel-to-gel variability and to enhance the resolution of age-related changes within each tissue. In these comparisons, the Rab24 levels were normalized to the level observed in the 1-day-old sample in each tissue, because comparative analysis between the different blots was not necessary. Rab24 levels in the brain significantly increased starting at 14 days of age, with a 4-5-fold increase compared to the 1-day and 7-day-old samples (Fig. 2 a). The elevated level was sustained in the 1-month, 3-month, 6-month and 9-month-old brain, suggesting that Rab24 may play a role in supporting brain function during later stages of development and aging. In contrast to the brain, Rab24 levels in the heart and skeletal muscle peaked during early postnatal development (1–14 days), followed by a significant reduction in adult tissues (Fig. 2 b, c). Rab24 levels in the pancreas and liver also displayed a notable pattern according to age. In both organs, Rab24 levels were initially lower in 1-day-old tissue, rose significantly by 7 days in the pancreas and by 14 days in the liver, and then decreased at 1 month of age, and stayed at the lower level in the 3- and 9-month samples (Fig. 2 d, e). In contrast to the dynamic changes seen in the tissues mentioned above, Rab24 levels in the kidney remained stable across all age groups (Fig. 2 f). Rab24 levels in the spleen and lung also showed less variability according to age (Fig. 2 g, h). Collectively, these findings highlight distinct patterns of Rab24 levels between different tissues, and according to age in each tissue, indicating the existence of tissue-specific regulatory mechanisms of Rab24 protein levels. In the brain, Rab24 levels increased after early development and remained elevated, suggesting a sustained role in postnatal housekeeping. Conversely, in the heart, muscle, pancreas, and liver, higher Rab24 level was linked to early developmental stages, decreasing as the tissues matured. Finally, the kidney, spleen, and lung exhibited more stable Rab24 levels across all tested age groups. In the brain, Rab24 is predominantly localized to neurons Given that the brain exhibited the highest levels of Rab24 among all tested organs, we next investigated Rab24 localization across different brain regions and cell types using immunohistochemistry (IHC). To explore potential changes in staining with age, we analysed brain tissue from mice ranging from 7 days to 6 months of age. Based on the morphology of the cells with a positive IHC signal, Rab24 was predominantly localized to neurons in various brain regions. Notably, Purkinje cells in the cerebellum exhibited Rab24 staining across all examined age groups, from postnatal day 7 to 6-months, with minimal age-related changes in staining intensity (Fig. 3 a-d). In contrast, Rab24 staining was either absent or low in many neuronal populations of 7-day-old mice (Fig. S3). However, in older mice (1, 3, and 6 months), Rab24 levels increased in many types of neurons in different brain regions, consistent with our western blot data (Fig. 4 ). Region-specific analysis revealed Rab24-positive neurons throughout the thalamus (Fig. 4 a, c). Within the hippocampus, weak to moderate Rab24 staining was detected in neurons of the dentate gyrus, as well as in the CA1 and CA3-CA4 subfields (Fig. 4 a, d-f). In the isocortex, Rab24 staining was observed in neurons distributed across the entire cortical mantle (Fig. 4 a, g). In the midbrain, Rab24 was prominently stained in neurons of several sites, including the substantia nigra, red nucleus, mesencephalic trigeminal nucleus and interpeduncular nucleus (Fig. 4 a, b, h-k). Similarly, in the hindbrain, Rab24 staining was high in neurons of the trigeminal motor nucleus, pontine grey, and reticular nucleus of the pons (Fig. 4 a, b, l-n), and low to moderate in neurons of the cochlear nucleus (Fig. 4 b, o). Rab24 staining in different types of epithelial cells IHC analysis revealed Rab24 staining in epithelial cells across multiple organs, with notable age-dependent differences in staining patterns. While 7-day-old mice exhibited somewhat varying epithelial staining patterns, tissues of older mice (1, 3, and 6 months) displayed more consistent staining intensities. Representative images from 7-day-old and 1-month-old mice show the age-related differences in epithelial Rab24 staining (Fig. 5 a-h). Ependymal cells line the ventricles in the brain. In the ependymal layer, Rab24 staining was stronger compared to the surrounding brain tissue in 7-day-old mice (Fig. 5 a). However, in older mice, Rab24 staining in the ependyma was more comparable to that of the adjacent tissue (Fig. 5 b). In the kidney, Rab24 staining followed a similar pattern in younger and older animals, with higher staining in the epithelial cells of the proximal convoluted tubules compared to the distal tubules and glomeruli (Fig. 5 c, d). The less differentiated tubules showed weaker Rab24 staining in the 7-day-old kidney (Fig. 5 c). In 1-month-old kidney, Rab24 staining was slightly stronger in the proximal tubules than in the distal tubules. Additionally, Rab24 staining was also observed in the parietal cells of the Bowman’s capsule (Fig. 5 d). In the lungs, Rab24 staining was prominent in the bronchiolar epithelium across all age groups, but differences were observed in the alveolar lining between young and old tissue. In 7-day-old mice, pneumocytes lining the alveolar walls showed stronger Rab24 staining compared to older animals (Fig. 5 e, f). Despite this age-related decrease, the bronchiolar epithelial cells maintained consistently high Rab24 levels throughout all age groups. In the exocrine pancreas, Rab24 staining was notably stronger in the acinar epithelial cells of 7-day-old mice compared to older animals (Fig. 5 g, h). The endocrine cells of the islets of Langerhans consistently showed stronger Rab24 staining compared to the surrounding exocrine tissue across all age groups (Fig. 5 g, h). Finally, the multicancer TMAs included also normal tissues from some organs, which enabled comparisons of the Rab24 staining in mouse tissues with the corresponding human adult tissues. Analysis of the human kidney, lung and pancreas revealed RAB24 staining patterns similar to those observed in adult mouse tissues (Fig. S4a-d). Rab24 staining in the heart, skeletal muscle, liver and spleen In the heart, Rab24 staining was moderate in cardiomyocytes of 7-day-old animals with markedly reduced staining in older mice (1, 3, and 6 months, Fig. 6 a, b). In the older mice, both cardiomyocytes and cardiac endothelial cells showed low Rab24 staining (Fig. 6 b). A similar but less prominent age-dependent decrease in Rab24 staining was observed in the skeletal muscle (Fig. 6 c, d). In contrast, Rab24 staining in the liver remained low in all age groups included in the IHC analysis (Fig. 6 e, f). In the spleen, Rab24 staining was moderate in both 7-day-old and older animals, with uniform staining across the white and red pulp (Fig. 6 g, h). Spleen macrophages were weakly-stained in 7-day-old tissue, and moderately stained in 1-month old and older tissue. Analysis of human adult tissues from the skeletal muscle and liver revealed RAB24 staining patterns similar to those observed in the adult mouse tissues (Fig. S4e, f). RAB24 levels in human cancers RAB24’s known involvement in autophagy (Yla-Anttila, et al., 2015 ), endocytosis (Amaya, et al., 2016 ) and mitosis (Militello, et al., 2013 ), processes frequently dysregulated in cancer, suggests that its altered expression may contribute to malignant transformation and tumour progression. Given the dynamic and tissue-specific levels of mouse Rab24 observed during postnatal development and aging, we sought to investigate whether RAB24 levels are altered in cancer. To address this, we analysed RAB24 levels by IHC in 75 tumour types originating from 220 patients using tissue microarrays (TMAs) (Table S1 ). RAB24 staining intensity was quantified using the H-scores (Fig. 7 a-f), which were calculated with the QuPath software (Bankhead, et al., 2017 ). The H-score is a semi-quantitative metric that combines staining intensity and the percentage of positively stained cells, providing a standardised method for comparing staining intensities in tissue samples. Frequency distribution of the H-scores revealed that RAB24 staining in the cancers included in the TMAs predominantly fall within the moderate range, with 65% of samples exhibiting H-scores between 100 and 199, while the maximum is 300 (Fig. 7 g). Strong RAB24 staining (H-score ≥ 200) was observed in 30.6% of the cancers, while low staining (H-score ≤ 99) was identified in the remaining 4.4% (Fig. 7 g). In comparison, normal tissues removed alongside the tumours during surgery exhibited a more balanced distribution: 30.7% had lower RAB24 staining, 55.4% moderate staining, and 13.9% high staining (Fig. S5a). To provide an overview, we summarized the findings for all 75 cancer types in a table that includes their tissue of origin, average RAB24 H-scores in malignant and normal tissues, the difference of H-scores between cancer tissue and normal tissue, and the number of patients per cancer (Table S1 ). The data reveal distinct patterns in RAB24 levels across cancers. A majority of cancer types demonstrated elevated RAB24 levels in malignant tissues compared to normal tissues, including several breast and skin cancer subtypes (Table S1 , Fig. 7 h). Conversely, 24% of cancers, particularly those of the digestive system (e.g., pancreas, stomach, appendix) and urinary tract, showed reduced RAB24 levels in malignant tissues compared to normal tissues (Table S1 , Fig. 7 h). A subset of cancers showed negligible changes in RAB24 levels (difference in H-score below 20) between malignant and normal tissues (Table S1 ). For further analysis, the 75 cancer types were categorized into 21 groups based on tissue or organ of origin (Table S1 , Fig. S5b). This categorization facilitated cross-comparison of RAB24 levels across diverse cancer types. For example, cancers of the thyroid, skin and pancreas displayed high RAB24 staining, while cancers of the esophagus, soft tissue and stomach showed lower staining levels (Fig. S5b). Among these cancers, only soft tissue sarcomas exhibited a statistically significant increase in RAB24 staining in malignant versus normal tissues (Fig. 7 i). Other cancers demonstrated non-significant trends of increased RAB24 staining in malignant tissue (Fig. S5c-h), higher levels in normal tissues (Fig. S5i-k), or no substantial difference between normal and cancer tissue (Fig. S5l-n). RAB24 levels in pancreatic neuroendocrine tumours The expression level of RAB24 was reported to be decreased in patient samples and cell lines of pancreatic adenocarcinoma, which originates from the exocrine cells. RAB24 was also observed to be an independent low-risk factor in this cancer type (Deng, et al., 2022 , Yu, et al., 2021 ). These findings motivated us to investigate RAB24 protein levels in pancreatic neuroendocrine tumours (PNETs), which originate from the endocrine pancreatic islet cells. We analysed RAB24 protein levels using TMAs containing samples from 120 patients with PNET. The TMAs contained tissue samples from three regions: the tumour, the tumour edge, and normal pancreatic tissue (Fig. 8 a-c). IHC staining for RAB24 was performed, and the H-scores were quantified using QuPath software. In tumour and tumour edge samples, the H-score was calculated separately for tumour cells and connective tissue, while in normal tissue samples, the H-score was determined for islet cells (the tissue of origin for PNETs) and connective tissue. The analysis revealed significantly higher RAB24 staining in pancreatic islet cells from normal tissue compared to the tumour and tumour edge samples (Fig. 8 d). No difference in RAB24 levels was observed between tumour and tumour edge samples. Furthermore, in connective tissue, RAB24 staining was significantly lower than in tumour cells. In normal tissue, RAB24 staining was lower in connective tissue compared to islet cells (Fig. 8 d). Notably, no differences in RAB24 levels were detected between connective tissue in tumour, tumour edge, and normal tissue (Fig. 8 d). To determine whether RAB24 protein levels vary with tumour grade, we performed IHC staining for Ki-67, a well-established proliferation marker (Fig. S6). Based on Ki-67 indices (Nagtegaal, et al., 2020 ), the majority of PNET samples (92.2%) were classified as low-grade (G1, 0–3% Ki-67-positive cells), while 6% were classified as intermediate-grade (G2, 3–20% Ki-67-positive cells), and 1.7% as high-grade (G3, more than 20% Ki-67-positive cells). Quantitative analysis of RAB24 H-scores revealed a significant decrease in RAB24 expression in higher-grade tumours, with PNET G1 samples displaying higher RAB24 levels compared to G2 and G3 tumours (Fig. 8 e). Discussion Our study provides a comprehensive analysis of Rab24 levels in mouse tissues from early postnatal age to adulthood, revealing distinct tissue-specific patterns and dynamic changes with age. We also analysed RAB24 levels in human cancers in comparison to peritumoral tissues, and found disease-specific alterations in Rab24 levels in the tumours. Age- and tissue-specific changes in Rab24 level in mouse tissues Rab24 levels in several tissues underwent dynamic changes during early postnatal development, followed by stabilization in adulthood. These findings indicate that Rab24 may contribute to cellular processes during organ development, with a potential role in tissue homeostasis in maturity. While our data do not provide direct evidence that Rab24 is critical for these processes, previous studies have demonstrated its essential role in Purkinje neuron survival (Agler, et al., 2014). In the brain, Rab24 levels increased significantly between 7 and 14 days of age and remained elevated into adulthood. Immunohistochemistry showed that different types of neurons have the highest Rab24 levels. The significant increase in brain Rab24 level coincides with the peak of synaptogenesis, a period marked by heightened neuronal activity and metabolic demands (Faria-Pereira and Morais, 2022, Li, et al., 2010). Interestingly, Rab24 has been found to localize to synaptic vesicles (Taoufiq, et al., 2020). These findings suggest a possible role for Rab24 in neuronal differentiation and long-term neuronal maintenance. In addition to synaptogenesis, also other processes are activated in the mouse brain during postnatal days 1-10, including production, migration and differentiation of additional neurons, and selective elimination of unnecessary neurons by programmed cell death (Chen, et al., 2017a). Within the brain, immunohistochemistry confirmed robust Rab24 levels in different types of neurons, including neurons involved in sensory processing, motor control and coordination, such as Purkinje cells. Interestingly, Rab24 protein staining in Purkinje cells was already evident at 7 days of age, and stayed similar until adulthood. This aligns with prior evidence showing that a Rab24 mutation leads to Purkinje cell degeneration in dogs (Agler, et al., 2014), underscoring the essential role of Rab24 in maintaining this specialized neuronal population. In several peripheral tissues, including the heart, skeletal muscle, pancreas and liver, Rab24 levels decreased after early postnatal development, i.e., after 14 days of age. The elevated Rab24 levels coincide with a time of increased biosynthetic and metabolic activity associated with organ maturation. In the heart and skeletal muscle, Rab24 levels peaked between 1 and 14 days of age, a period of rapid cellular growth and differentiation (Gattazzo, et al., 2020, Li, et al., 1996), before decreasing to stable levels in adulthood. The initially higher levels of Rab24 in the heart coincide with a period of rapid growth and cellular differentiation of cardiomyocytes (Piquereau, et al., 2010). Similarly, Rab24 level in the liver and pancreas was notably elevated at 7 and 14 days (pancreas) or 14 days (liver) of age compared to older tissues, correlating with the high metabolic and biosynthetic demands of these tissues during postnatal development (Bonner-Weir, et al., 2016, Liang, et al., 2022). Notably, Rab24 level in the pancreatic islets of Langerhans remained consistently higher than in the surrounding exocrine tissue across all ages, suggesting a sustained role for Rab24 in the hormone-secreting cells. In the lung, Rab24 staining was elevated in both the alveolar lining cells and the bronchiolar epithelium of 7-day-old mice. The alveolar lining appeared denser in the young animals, with stronger Rab24 staining compared to older mice. Active alveolar remodelling and expansion during early postnatal development coincide with the 7-day sample collection (Negretti, et al., 2021). In contrast, Rab24 staining in the club cells of the bronchiolar epithelium remained consistently high across all ages. These cells synthesise and secrete the lining fluid of the respiratory epithelium (Blackburn, et al., 2023). Organs such as the kidney and spleen displayed relatively stable Rab24 levels across all age groups, suggesting a more consistent demand in Rab24 protein. In the kidney, Rab24 was uniformly stained in the proximal convoluted tubular epithelial cells. Greater variability in Rab24 staining was observed in the kidney tubules of 7-day-old mice. Stronger staining was observed particularly in larger, more differentiated tubules, suggesting a role for Rab24 during tubular differentiation or function. Rab24 was also stained in the parietal epithelial cells of the Bowman’s capsule but not in podocytes of the glomerulus. In the spleen, Rab24 was stained in both the white and the red pulp. Within the red pulp of 1-month-old and older animals, Rab24 was enriched in large cells, likely macrophages, which are critical for immune surveillance and phagocytosis. The differences in Rab24 levels between 7 and 14-day-old and older animals suggest that Rab24 is developmentally regulated. Because pathways such as autophagy and endocytosis, where Rab24 has documented roles (Amaya, et al., 2016, Yla-Anttila, et al., 2015), are essential for both differentiation and maintenance, Rab24 may contribute to these processes throughout life rather than having distinct functions at different stages. During early postnatal development, Rab24 may contribute to neuronal differentiation and/or maintenance, epithelial remodelling, and organ maturation. In adulthood, the role of Rab24 may shift towards maintenance of homeostasis by regulating similar mechanisms. Rab24 staining in epithelial cells suggests a potential role in the biology of this cell type. Epithelial cells undergo continuous turnover and require tightly regulated intracellular trafficking processes, including endocytosis and autophagy, to maintain tissue integrity (Schwarz, et al., 2007, Wong, et al., 2019). Many epithelial cells have also active secretory functions: ependymal cells secrete the cerebrospinal fluid, and pancreatic islet cells secrete hormones. Epithelial cells can also be active in endocytosis, like the kidney proximal tubule epithelium. In developing tissues, Rab24 could contribute to epithelial remodelling and differentiation, while in mature tissues, it may support homeostatic processes such as barrier maintenance and cellular renewal. Further studies are needed to define the precise contributions of Rab24 to epithelial cell functions as well as age and tissue-specific roles. Insights from the mouse data highlight the developmental regulation of Rab24, with levels peaking during critical periods of organ development in the brain, heart, muscle, pancreas and liver. The age-dependent Rab24 levels suggest that Rab24 levels may also vary during different stages of cancer progression, similar to the surge observed in early postnatal development. Rab24 levels in cancers The dynamic changes in Rab24 levels observed during postnatal development in mice, particularly surges during periods of organ maturation, may mirror mechanisms exploited or disrupted during tumorigenesis. For instance, the processes governing Rab24’s involvement in endocytosis, autophagy, as well as mitosis, cell migration and invasion could be exploited by cancer cells to support their growth, survival, and metastasis (Amaya, et al., 2016, Chen, et al., 2017b, Militello, et al., 2013, Yla-Anttila, et al., 2015). Furthermore, the tissue-specific dynamic variations in Rab24 levels with age emphasize the importance of context in the possible roles of Rab24. Dysregulation in cancer may arise from perturbations in the finely-tuned developmental programs. Our results showed that RAB24 levels in cancers of the breast and skin were higher than in the corresponding normal tissues, while the opposite was found in cancers of the digestive system and the urinary tract. The observed heterogeneity in RAB24 levels across various cancer types aligns with its multifaceted roles in cellular processes, which may be hijacked or altered in cancer cells to support oncogenic processes. For example, elevated RAB24 levels in HCC and HCC cell lines have been associated with enhanced growth and metastasis (He, et al., 2002, Yang, et al., 2020, Zhang, et al., 2020, Zhu, et al., 2020). This elevation reflects the role of RAB24 in promoting malignant phenotypes such as epithelial-mesenchymal transition (EMT), vasculogenic mimicry, and cell adhesion (Chen, et al., 2017b). RAB24 is also an unfavourable prognostic marker in prostate cancer (Hu, et al., 2020). Conversely, reduced RAB24 expression in other cancers may indicate downregulation in favour of alternative survival mechanisms, reflecting the adaptability of cancer cells in diverse tissue environments. Our findings revealed that RAB24 levels are significantly reduced in pancreatic neuroendocrine tumours (PNETs) compared to normal pancreatic islet cells. Given the known involvement of RAB24 in autophagy and endocytosis, the downregulation in PNETs may reflect alterations in pathways that contribute to tumour progression. Notably, while RAB24 staining was consistently lower in tumour cells than in normal islet cells, H-scores in malignant tissues exhibited substantial variability among patients, with values varying between 294.1 and 129.6. To investigate whether this variability correlated with tumour grades, we assessed the tumour grades using Ki-67 staining. Our analysis revealed an inverse correlation between RAB24 level and tumour grade, further supporting a potential role of RAB24 in PNET biology. PNETs classified as G1 exhibited significantly higher RAB24 levels than those classified as G2 or G3. Although the number of G2 and G3 samples in our cohort was limited, our findings suggest that RAB24 downregulation may be linked to more aggressive tumour phenotypes. Given that Ki-67 is a well-established marker of cell proliferation, the inverse relationship between Ki-67 and RAB24 staining suggests that RAB24 downregulation may be associated with increased proliferative capacity and tumour aggressiveness in PNETs. Future studies with larger patient cohorts should explore whether RAB24 levels correlate with clinical parameters such as patient survival or response to therapy, and whether loss of RAB24 contributes functionally to increased malignancy. Collectively, our findings provide a comprehensive overview of RAB24 levels across a wide spectrum of human cancers. The observed differences in RAB24 levels between malignant and normal tissues, as well as among cancer types, suggest that RAB24 may play context-dependent roles in malignancy. These insights lay the groundwork for future studies to elucidate RAB24’s functional contributions to tumorigenesis and its potential as a diagnostic tool or therapeutic target in cancer. Conclusions and implications In this study, tissue and age-specific levels of Rab24 in mouse were observed by western blot and confirmed in-situ by IHC. The dynamic changes in Rab24 levels during early development suggest roles in organ maturation in certain tissues including the brain, heart, skeletal muscle, pancreas and liver while the stable levels in adulthood point to maintenance functions. Further, our results showed that RAB24 levels in cancers of the breast and skin were higher than in the corresponding normal tissues, while the opposite was found in cancers of the digestive system and the urinary tract. In pancreatic PNET, RAB24 levels were lower than in normal islet cells. Future research is needed on possible contributions of Rab24 to age-related disorders and on its potential as a biomarker or therapeutic target in cancer and other diseases. Further studies of the molecular mechanisms of Rab24 in health and disease will be pivotal in harnessing its diagnostic and therapeutic potential. Declarations Acknowledgements We thank the core facilities at the Institute of Biomedicine, University of Turku: Histology core facility for assisting with the sectioning of paraffin-embedded mouse tissues, and Medisiina Imaging Center for availability of the slide scanner. Hira Javed is also thanked for assistance in paraffin sectioning. We thank Kati Kuipers and the Finnish Center for Laboratory Animal Pathology, University of Helsinki, for the Ki-67 staining of the PNET sections. Funding This project was funded by Marie Skłodowska-Curie ETN grant under the European Union’s Horizon 2020 Research and Innovation Programme (Grant Agreement No 765912, DRIVE), the Institute of Biomedicine, University of Turku, Magnus Ehrnrooth Foundation (March 6, 2021), and the Research Council of Finland (grant No 351215). H.G.M.R was supported by the Finnish Cultural Foundation Central Fund (grant No 00220857) and the Turku University Foundation (grant No 081769). P.S. was supported by RESET (Resilient and Just Systems) research profiling funding from the Research Council of Finland (PROFI7 2023–2028). Competing interests The authors declare no competing interests. References Agler C, Nielsen DM, Urkasemsin G, Singleton A, Tonomura N, Sigurdsson S, Tang R, Linder K, Arepalli S, Hernandez D, Lindblad-Toh K, van de Leemput J, Motsinger-Reif A, O'Brien DP, Bell J, Harris T, Steinberg S, Olby NJ (2014) Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24. Plos Genet 10:e1003991 Amaya C, Militello RD, Calligaris SD, Colombo MI (2016) Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation. 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Aging 12:14582–14592 Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx Table S1 List of cancer samples included in the multicancer TMAs used in this study, including the H-scores for the intensity of RAB24 immunohistochemical staining. FigureS1.tif Fig. S1 Immunohistochemical staining with and without Rab24 antibody in various mouse tissues. The two staining protocols were performed using consecutive serial sections. Representative images are shown for anti-Rab24 and the corresponding control sections, as indicated on the left. Positive Rab24 staining is visible as brown colour in the anti-Rab24-stained sections. Scale bars: 50 µm. FigureS2.tif Fig. S2 Immunohistochemical staining of RAB24 in TMAs containing samples of human pancreatic neuroendocrine tumours and pancreatic normal tissue. Representative images are shown for anti-RAB24 staining (a, b) and the serial control section stained without the primary antibody (c, d). RAB24 staining is visible as brown colour in the anti-RAB24-stained tissue cores. Scale bars: 2 mm (a, c) and 200 µm (b, d). FigureS3.tif Fig. S3 Rab24 immunohistochemical staining in the hippocampus, midbrain and hindbrain of 7-day-old mice. The boxed areas in panels a and b indicate the regions shown at higher magnification in panels c-j. The brown colour indicates Rab24 staining. Scale bar: 1 mm (a, b) and 50 µm (c-j). FigureS4.tif Fig. S4 RAB24 immunohistochemical staining in adult human tissues. Arrowheads indicate RAB24-positive epithelial cells lining the kidney tubular cells in panel a, and the bronchiolar epithelium in panel b. G, kidney glomerulus; B, bronchiole lumen; I, pancreatic islet. Scale bar: 50 µm. FigureS5.tif Fig. S5 Comparison of the H-scores for RAB24 immunohistochemical staining intensities in malignant and normal tissues grouped by organ or tissue of origin.(a) Frequency distribution of RAB24 H-scores in normal tissues removed together with tumour samples during surgery. (b) Overview of RAB24 H-scores across 21 categories of cancers grouped by tissue or organ of origin. The horizontal line indicates the average. (c–n) Tissue categories demonstrating a trend of increased (c-h), decreased (i-k), or of no change (l-n) in RAB24 H-score in malignant versus normal tissues. The horizontal lines indicate mean ± SEM. Statistical significance was determined by Wilcoxon test; none of the differences between malignant and normal tissue in panels c-n is statistically significant. FigureS6.tif Fig. S6 Representative immunohistochemical staining of Ki-67 in PNET samples, illustrating tumour grade classification based on Ki-67 index. G1, 0-3% Ki-67-positive cells; G2, 3-20% Ki-67-positive cells; and G3, more than 20% Ki-67-positive cells. Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Cell and Tissue Research → Version 1 posted Editorial decision: Revision requested 22 Apr, 2025 Reviews received at journal 04 Apr, 2025 Reviews received at journal 02 Apr, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 19 Mar, 2025 Reviewers agreed at journal 16 Mar, 2025 Reviewers invited by journal 13 Mar, 2025 Editor assigned by journal 11 Mar, 2025 Submission checks completed at journal 11 Mar, 2025 First submitted to journal 07 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6176716","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431285238,"identity":"dd99f456-8729-4cfa-b07e-193c90cec156","order_by":0,"name":"H. G. 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(b-h) Rab24 protein levels were analysed using western blotting with total protein staining (TPS) as the loading control. In order to compare Rab24 levels between different blots, tissue extract from one 1-month-old liver was used as a control sample in all blots. The Rab24 signals normalized to TPS were further normalized to the control in each blot. Representative western blot images and quantification of\u003cstrong\u003e \u003c/strong\u003eRab24 level in the indicated organs at each age group are shown. The graphs show the mean and standard deviation calculated from four mice in each age group. Kruskal-Wallis test and post hoc Dunn's multiple comparisons test were used for statistical significance: * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/0d9df1cae7d9a235ce7ff385.png"},{"id":79065180,"identity":"7ad62a5e-e188-47ee-a5ea-e5949dfee5b8","added_by":"auto","created_at":"2025-03-24 04:02:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1600550,"visible":true,"origin":"","legend":"\u003cp\u003eRab24 levels in mouse tissues according to age.\u003cstrong\u003e \u003c/strong\u003eRab24 protein levels were examined using western blotting with total protein staining (TPS) as the loading control. The Rab24 signals normalized to TPS were normalized to the 1-day old sample in each blot. (a-h) Representative western blot images and quantification of Rab24 levels for each tissue are shown. The graphs show the mean and standard deviation calculated from four mice for each age group. Mann-Whitney test was used for statistical significance of adjacent ages: * p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/4d88116a3dbc21a8fc0fb7c9.png"},{"id":79065164,"identity":"1f4e3d89-ab5f-4994-b584-4ae8c0688b27","added_by":"auto","created_at":"2025-03-24 04:02:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2714777,"visible":true,"origin":"","legend":"\u003cp\u003eRab24 immunohistochemical staining in the murine cerebellum according to age.\u003cstrong\u003e \u003c/strong\u003eRepresentative images showing the cerebellum of mice aged 7 days to 6 months.\u003cstrong\u003e \u003c/strong\u003eArrowheads indicate Rab24-positive Purkinje cells. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/8cc3fde83de69741261fb591.png"},{"id":79065170,"identity":"7e93902e-b11b-4460-b25a-e1bda77364df","added_by":"auto","created_at":"2025-03-24 04:02:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9578249,"visible":true,"origin":"","legend":"\u003cp\u003eRab24 immunohistochemical staining in the 1-month-old mouse brain.\u003cstrong\u003e \u003c/strong\u003e(a, b) Low-magnification images showing the hippocampus, midbrain and hindbrain regions.\u003cstrong\u003e \u003c/strong\u003eThe boxed areas in panels a and b indicate the regions shown at higher magnification in panels c-o. The brown Rab24-positive cells are neurons.\u003cstrong\u003e \u003c/strong\u003eScale bar: 1 mm (a, b) and 50 μm (c-o).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/afd64ddd5b919a2eba9a9860.png"},{"id":79065171,"identity":"d8bc36c8-8464-4756-9a5d-3a4a276c3cb0","added_by":"auto","created_at":"2025-03-24 04:02:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4887635,"visible":true,"origin":"","legend":"\u003cp\u003eRab24 immunohistochemical staining in epithelial cells in mouse tissues.\u003cstrong\u003e \u003c/strong\u003eRepresentative images showing Rab24 staining of 7-day-old (a, c, e, g) and 1-month old (b, d, f, h) mice. Ependyma lining the brain ventricles (a, b), kidney (c, d), lung (e, f), and pancreas (g, h) are shown. Arrowheads indicate Rab24-positive epithelial cells. G, kidney glomerulus; A, alveolus; B, bronchiole; I, pancreatic islet. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/5a40ccc51a46563f897b5c66.png"},{"id":79065174,"identity":"bc1e62e3-454a-4591-bac3-49571a640c4a","added_by":"auto","created_at":"2025-03-24 04:02:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5194975,"visible":true,"origin":"","legend":"\u003cp\u003eAge-dependent Rab24 immunohistochemical staining in the heart, skeletal muscle, liver, and spleen. Representative images showing 7-day-old (a, c, e, g) and 1-month old (b, d, f, h) tissues. The heart (a, b), skeletal muscle (c, d), liver (e, f), and spleen (g, h) are shown. (g, h) Insets and arrowheads indicate macrophages in the spleen, showing low Rab24 staining at 7 days, and moderate Rab24 staining at 1 month. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/2cbf10900114ef99d9de233e.png"},{"id":79065173,"identity":"d9763ed8-60c9-4b37-8b0d-958200828557","added_by":"auto","created_at":"2025-03-24 04:02:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4648532,"visible":true,"origin":"","legend":"\u003cp\u003eRAB24 immunohistochemical staining intensities across 75 cancer types analysed in tissue microarrays (TMAs). (a–f) Representative images showing RAB24 staining in malignant tissues exhibiting different RAB24 staining intensity and H-scores calculated using QuPath software. Scale bar: 50 µm. (g) Frequency distribution of RAB24 H-scores in the 75 cancer types. (h) Waterfall plot showing the top 10 cancer types with increased and decrease H-scores in malignant tissue compared with the corresponding normal tissue. Abbreviations: Breast ER+, ductal carcinoma of the breast, estrogen-receptor positive; FTC, follicular carcinoma of the thyroid gland; Breast HER2+, ductal carcinoma of the breast, human epidermal growth factor receptor 2-positive; TSCC, tonsillar squamous cell carcinoma; cSCC, cutaneous squamous cell carcinoma; CAC, cervical adenocarcinoma; Rectal NEC, rectal neuroendocrine carcinoma; Breast, ductal carcinoma of the breast, unspecified; PNET G1, pancreatic neuroendocrine tumour, low-grade (G1); ANEC, appendiceal neuroendocrine carcinoma; GAC, gastric adenocarcinoma; A-NET G2, appendiceal neuroendocrine tumour, intermediate grade (G2); PNEC G3, pancreatic neuroendocrine carcinoma G3; pRCC, papillary renal cell carcinoma; CCA, cholangiocarcinoma; A-NET G1, appendiceal neuroendocrine tumour, low-grade (G1); PAC, pancreatic adenocarcinoma; dGAC, diffuse gastric adenocarcinoma. (i) Paired dot plot of RAB24 levels in soft tissue cancers showing a statistically significant increase in RAB24 staining in malignant versus normal tissues. The horizontal lines indicate mean ± SEM. Statistical significance was determined by Wilcoxon test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/40b62841deb4ddc5a25f5c30.png"},{"id":79066037,"identity":"39ff4dbe-f78f-4e7b-ac47-025be62c5896","added_by":"auto","created_at":"2025-03-24 04:18:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3225613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRAB24 immunohistochemical staining in pancreatic neuroendocrine tumours (PNETs) and normal pancreatic tissue \u003c/strong\u003eanalysed in tissue microarrays (TMAs)\u003cstrong\u003e.\u003c/strong\u003e (a–c) Representative images of RAB24 staining in (a) tumour tissue, (b) tumour edge, and (c) normal pancreatic tissue. The tissues in the representative images showed H-scores close to the mean H-score of the respective tissue type. Connective tissue (C), tumour (T), acinar tissue (A), and pancreatic islet (I) are indicated. Scale bar: 50 µm. (d, e) Quantification of RAB24 staining intensities. H-scores were determined using QuPath software for tumour cells, islet cells, and connective tissue (d). \u0026nbsp;RAB24 H-scores in low-grade (G1) and intermediate (G2) and high-grade (G3) PNET samples (e). Individual data points and mean ± SEM are shown, with statistical significance determined by Kruskal-Wallis test and post-hoc Dunn’s multiple comparison test (d), and Mann-Whitney test (e).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/ca890e4865ad0c260cd8403c.png"},{"id":101262053,"identity":"644cabc2-9c56-4a5c-8c38-babd0daf9e4c","added_by":"auto","created_at":"2026-01-27 21:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31940641,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/e0029a18-8d24-49db-aeb1-7a82e61d4c60.pdf"},{"id":79065161,"identity":"7771df9f-142a-4db9-a838-f7b97f2589ad","added_by":"auto","created_at":"2025-03-24 04:02:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eList of cancer samples included in the multicancer TMAs used in this study, including the H-scores for the intensity of RAB24 immunohistochemical staining.\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/13cf23b01f9b7ab0f8f0233b.xlsx"},{"id":79065190,"identity":"b5c8fc82-625e-40d5-a68e-cfc05eee5aaf","added_by":"auto","created_at":"2025-03-24 04:02:35","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30225358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 \u003c/strong\u003eImmunohistochemical staining with and without Rab24 antibody in various mouse tissues. The two staining protocols were performed using consecutive serial sections. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRepresentative images are shown for anti-Rab24 and the corresponding control sections, as indicated on the left. Positive Rab24 staining is visible as brown colour in the anti-Rab24-stained sections. Scale bars: 50 µm.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/8622f41ed52092527d98f41a.tif"},{"id":79065196,"identity":"6c5f83dd-459e-4ca6-97ec-afd4a1e506d8","added_by":"auto","created_at":"2025-03-24 04:02:35","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30582570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 \u003c/strong\u003eImmunohistochemical staining of RAB24 in TMAs containing samples of human pancreatic neuroendocrine tumours and pancreatic normal tissue. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRepresentative images are shown for anti-RAB24 staining (a, b) and the serial control section stained without the primary antibody (c, d). RAB24 staining is visible as brown colour in the anti-RAB24-stained tissue cores. Scale bars: 2 mm (a, c) and 200 µm (b, d).\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/cbeb96712a55a56c13c79204.tif"},{"id":79066038,"identity":"b1e679d5-8ffb-40f0-ac90-4f6923d5ea97","added_by":"auto","created_at":"2025-03-24 04:18:36","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":30149574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3 \u003c/strong\u003eRab24 immunohistochemical staining in the hippocampus, midbrain and hindbrain of 7-day-old mice.\u003cstrong\u003e \u003c/strong\u003eThe boxed areas in panels a and b indicate the regions shown at higher magnification in panels c-j. The brown colour indicates Rab24 staining. Scale bar: 1 mm (a, b) and 50 µm (c-j).\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/2e4b582fb4d3307085f2d8e0.tif"},{"id":79065206,"identity":"de96104f-f8b3-4ae9-8713-848c60bd78ba","added_by":"auto","created_at":"2025-03-24 04:02:36","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":30389842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4 \u003c/strong\u003eRAB24 immunohistochemical staining in adult human tissues. Arrowheads indicate RAB24-positive epithelial cells lining the kidney tubular cells in panel a, and the bronchiolar epithelium in panel b. G, kidney glomerulus; B, bronchiole lumen; I, pancreatic islet. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/3a52d0a596c76474bfef99cc.tif"},{"id":79065207,"identity":"ff4bfe49-114e-42b4-ba87-55c82c1d32fc","added_by":"auto","created_at":"2025-03-24 04:02:37","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":30055358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S5\u003c/strong\u003e \u003cstrong\u003eComparison of the H-scores for RAB24 immunohistochemical staining intensities in malignant and normal tissues grouped by organ or tissue of origin.\u003c/strong\u003e(a) Frequency distribution of RAB24 H-scores in normal tissues removed together with tumour samples during surgery. (b) Overview of RAB24 H-scores across 21 categories of cancers grouped by tissue or organ of origin. The horizontal line indicates the average. (c–n) Tissue categories demonstrating a trend of increased (c-h), decreased (i-k), or of no change (l-n) in RAB24 H-score in malignant versus normal tissues. The horizontal lines indicate mean ± SEM. Statistical significance was determined by Wilcoxon test; none of the differences between malignant and normal tissue in panels c-n is statistically significant.\u003c/p\u003e","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/da4743044cbdab50d7a658e7.tif"},{"id":79065210,"identity":"75a546d6-9add-4935-92cd-11da56e466ea","added_by":"auto","created_at":"2025-03-24 04:02:37","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":30127930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S6 \u003c/strong\u003eRepresentative immunohistochemical staining of Ki-67 in PNET samples, illustrating tumour grade classification based on Ki-67 index. G1, 0-3% Ki-67-positive cells; G2, 3-20% Ki-67-positive cells; and G3, more than 20% Ki-67-positive cells.\u003c/p\u003e","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6176716/v1/cc41ec40869d1e1b7655647e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rab24 protein levels show dynamic changes in mouse tissues and human cancers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRab GTPases regulate intracellular membrane trafficking events ranging from vesicle formation, vesicle transport, membrane tethering and membrane fusion. Rab24 was first described in 1993 to localize to the endoplasmic reticulum (ER), Golgi apparatus and late endosomes (Olkkonen, et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). It is an unusual member of the Rab family due to the presence of an atypical amino acid in the GTP-binding region (Erdman, et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe and others showed that Rab24 functions in autophagy (Munafo and Colombo, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Yla-Anttila, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), a catabolic process that recycles organelles and aggregate-prone proteins by transporting them to lysosomes, thereby producing substrates for biosynthesis and energy production. The Q38P point mutation in Rab24, leading to degeneration of cerebellar Purkinje neurons, was identified as the cause of canine hereditary ataxia in Gordon setters and Old English sheepdogs (Agler, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The affected canine neurons accumulate autolysosomes and ubiquitin-protein aggregates, in agreement with our results on the role of Rab24 in autophagy (Yla-Anttila, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Further, Rab24 was shown to regulate endosomal degradation by interacting with the late endosomal protein Rab7 (Amaya, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Together, these results show that Rab24 is important for neuronal health, possibly by regulating the delivery of autophagic and endocytic cargo to lysosomes.\u003c/p\u003e \u003cp\u003eIn humans, RAB24 has been associated with fatty liver disease and hepatocellular carcinoma. Liver RAB24 levels positively correlate with body fat and are highly increased in the livers of obese patients with non-alcoholic fatty liver disease (NAFLD) (Seitz, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Rab24 knockdown in mouse liver decreased hepatic fat and reduced serum cholesterol levels in obese mice, confirming the link between Rab24 levels and liver fat accumulation. Furthermore, several studies show RAB24 to be overexpressed, and/or be associated with, poor prognosis in liver cancer (Chen, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e, He, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Yang, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhang, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhu, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). RAB24 expression is increased in hepatocellular carcinoma (HCC) due to downregulation of miR-615-5p, which normally downregulates RAB24 expression (Chen, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). Ectopic overexpression of RAB24 facilitated HCC cell motility, invasion and adhesion, accelerated cell cycle progression, reduced apoptosis, and facilitated epithelial to mesenchymal transition, while knockdown of RAB24 had opposite effects in all these assays (Chen, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e). These data show that RAB24 plays a significant role in promoting the malignant phenotype of HCC cells. Furthermore, high RAB24 expression is an unfavourable prognostic marker in prostate cancer (Hu, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the contrary, RAB24 was reported to be an independent low-risk factor in pancreatic adenocarcinoma (Deng, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to the Human Protein Atlas (proteinatlas.org, Uhlen, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), RAB24 is classified as a potentially favourable marker in clear cell renal cell carcinoma and an unfavourable marker in glioblastoma. These findings highlight RAB24\u0026rsquo;s context-dependent prognostic value in different cancer types.\u003c/p\u003e \u003cp\u003eDespite these advances, the expression patterns of Rab24 in different tissues and developmental stages have not been analysed. Knowledge on these patterns can provide insights into Rab24\u0026rsquo;s possible physiological roles and potential contributions to age-related diseases. Given that mice are commonly used as model organisms in autophagy research, and that Rab24 plays a role in autophagy, the understanding of the normal age-related expression patterns of Rab24 protein is important, as such patterns may serve as potential confounding factors when interpreting experimental results. In this study, we analysed Rab24 protein levels using western blotting in the brain, heart, liver, lung, kidney, spleen, skeletal muscle, and pancreas of C57BL/6 mice in different age groups from postnatal days to 9 months. Immunohistochemistry was also performed in order to analyse which cell types in these tissues express Rab24 protein. Our results revealed distinct cell and tissue-specific patterns and dynamic, age-dependent changes in Rab24 levels. This is the first study to provide a comprehensive record of Rab24 protein levels in mouse tissues of different age. In addition, we analysed tissue microarrays of different human cancers, where RAB24 levels differ from the normal tissue. Our findings provide a basis for further studies regarding the role of RAB24 as a prognostic or predictive factor.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthical compliance\u003c/h2\u003e \u003cp\u003e All experimental procedures involving animals were ethically reviewed and approved by the Project Authorization Board of the Regional State Administrative Agency of Southern Finland (ESAVE/613/2019), and complied with the guidelines of the Directive 2010/63/EU of the European Union.\u003c/p\u003e \u003cp\u003e Sections from human cancer tissue microarrays (TMAs) were obtained from Helsinki Biobank, after the acceptance of the Ethics Committee of the Hospital District of Helsinki and Uusimaa (HUS/697/2020). The samples in the Helsinki Biobank are stored after receiving informed consent from all patients. The study conforms to the standards of the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of mouse tissue extracts and immunoblotting\u003c/h3\u003e\n\u003cp\u003eMice were group housed under controlled temperature and 12-h light-dark rhythm with same-sex littermates, and given free access to food and water. C57BL/6 mice of different age (1 day, 7 days, 14 days, 1 month, 3 months, 6 months, and 9 months) were sacrificed by cervical dislocation (1, 7 and 14-day-old animals) or by carbon dioxide (1 month and older animals). Four mice, two males and two females, were used for each age group. Samples from the cerebral cortex, heart, liver, lung, kidney, spleen, skeletal muscle (\u003cem\u003etibialis anterior\u003c/em\u003e) and pancreas were collected and snap-frozen in liquid nitrogen. For protein extraction, approximately 35 mg of tissue was homogenized in 140 \u0026micro;l of homogenization buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1% NP-40 and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (A32959, Thermo Scientific). After adding a 5-mm stainless steel bead (69989, Qiagen), the samples were lysed using a TissueLyser LT (85600, Qiagen) at 50 Hz for 3 min. Subsequently, an additional 140 \u0026micro;l of homogenization buffer was added, bringing the total buffer volume to 280 \u0026micro;l per 35 mg of tissue. The lysates were rotated end-over-end at +\u0026thinsp;4\u0026deg;C for 1 h and centrifuged at at +\u0026thinsp;4\u0026deg;C, 16,000 g for 15 min. Protein concentration of the supernatants was determined by bicinchoninic acid (BCA) assay (23228, Thermo Scientific), and SDS sample buffer (100 mM sodium phosphate, pH 7.5, 2% w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol, 0.004% w/v bromophenol blue) was then added. The samples were heated at +\u0026thinsp;95\u0026deg;C for 4 min, and stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003ePer lane, 10 \u0026micro;g of total protein were resolved on a 12% SDS-PAGE gel and blotted to a polyvinylidene difluoride (PVDF) membrane (88518, Thermo Scientific). Total proteins were stained using TotalStain Q (AC2225, Azure Biosystems) and the blots were imaged using Azure Sapphire imaging system (Azure Biosystems). For antibody staining, the membranes were blocked with 5% non-fat milk powder in Tris-buffered saline (100 mM Tris-HCl, pH 7.6, 1.5 M NaCl) containing 0.05% Tween-20 (TBS-T). Membranes were probed with affinity-purified rabbit anti-RAB24 (11445-1-AP, Proteintech) in blocking solution at +\u0026thinsp;4\u0026deg;C overnight. Membranes were washed, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (111-035-003, Jackson ImmunoResearch) at room temperature for 1 h, and the bands were visualized with Clarity\u0026trade; Western ECL Substrate (1705061, Bio-Rad). Blots were imaged using Azure Sapphire imaging system (Azure Biosystems), and the bands were quantified using Fiji/ImageJ (Schindelin, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In order to compare Rab24 levels between different blots, tissue extract from one 1-month-old liver was used as a control sample in the blots. The Rab24 signals were first normalized to total protein, and then normalized to the control sample in each gel.\u003c/p\u003e\n\u003ch3\u003ePreparation of mouse tissues for immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry was performed for mice aged 7 days and 1, 3 and 6 months. Cervical dislocation followed by decapitation was used for euthanisation of 7-day-old C57BL/6 mice. Older mice (1, 3 and 6 months) were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg), and transcardially perfused with phosphate-buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde (PFA) in PBS, using a peristaltic pump at a flowrate of 5 ml per minute. For all age groups, samples from the brain, heart, liver, lung, kidney, spleen, skeletal muscle and pancreas were collected and post-fixed in 4% PFA in PBS at +\u0026thinsp;4\u0026deg;C for 24 h. The samples were dehydrated, embedded in paraffin, and 5-\u0026micro;m sections were cut and mounted on glass slides.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eFor Rab24 staining, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidases were quenched in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 20 min. Antigen retrieval was performed in 10 mM citrate buffer, pH 6.0, by microwave heating at 640 W for 7 min and at 480 W for 7 min. Sections were permeabilized in 0.1% Triton X-100 in TBS-T for 5 min. Blocking was performed with 5% normal goat serum in TBS at room temperature for 1 h. Sections were incubated in affinity-purified rabbit anti-RAB24 (11445-1-AP, Proteintech) in 5% normal goat serum at +\u0026thinsp;4\u0026deg;C overnight. Subsequently, the sections were washed and incubated in biotinylated goat anti-rabbit IgG (PK-6101, Vector Laboratories) at room temperature for 1 h. Sections were washed, incubated with an avidin horseradish peroxidase complex (PK-6101, Vector Laboratories) in PBS at room temperature for 40 min, washed again and incubated in 3,3\u0026rsquo;-diaminobenzidine (DAB, ready-made reagent, SK-4100, Vector Laboratories) for 1 min. Slides were counterstained with Mayer\u0026rsquo;s haematoxylin (105.3 mM aluminium potassium sulphate, 3.308 mM haematoxylin, 505.3 \u0026micro;M sodium iodate, 4.758 mM citric acid, 302.2 mM chloral hydrate) for 1 min, dehydrated in a graded alcohol series and mounted using Pertex\u0026reg; mounting medium (00811, HistoLab).\u003c/p\u003e \u003cp\u003eTo confirm the specificity of the immunostaining, serial sections of the tissues were stained both with the protocol described above, and with a control protocol in which the primary antibody incubation was replaced by a prolonged blocking step (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The same control staining was done for the pancreatic neuroendocrine tumour sections (Fig. S2).\u003c/p\u003e \u003cp\u003eFor Ki-67 immunohistochemical staining, TMA sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed in 10 mM citrate buffer, pH 6.0, at +\u0026thinsp;99\u003csup\u003eo\u003c/sup\u003eC for 20 min in PT Module (Lab Vision\u003csup\u003e\u0026trade;\u003c/sup\u003e). Slides were set in Shandon\u003csup\u003e\u0026trade;\u003c/sup\u003e Coverplate system. Endogenous peroxidases were quenched in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS for 10 min, and blocking was performed with 20% normal goat serum in PBS at room temperature for 20 min. Sections were incubated in rabbit monoclonal anti-Ki-67 (# RM-9106-S1, Thermo Scientific) in 1% BSA in PBS at room temperature for 60 min. Subsequently, the sections were washed and incubated in biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories) in PBS at room temperature for 30 min. Sections were washed, incubated with an avidin-HRP complex (PK-6100, Vector Laboratories) in PBS at room temperature for 30 min, washed again and incubated in DAB (BS04, ImmunoLogic a WellMed Company) for 6 min. Slides were counterstained with Harris haematoxylin (1.09253, Sigma-Aldrich), dehydrated in a graded alcohol series and mounted using Pertex\u0026reg; mounting medium. Control slide was incubated without the primary antibody.\u003c/p\u003e \u003cp\u003eImages of whole slides were acquired using a Pannoramic 250 Flash slide scanner equipped with a 20x objective (3DHistech). Images were analysed and cropped using CaseViewer (3DHistech) and Fiji/ImageJ software (Schindelin, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAnalysis of human tissue microarrays\u003c/h3\u003e\n\u003cp\u003eParaffin sections from tissues microarrays (TMAs) containing samples from 75 different types of human cancers belonging to 220 patients, and from TMAs containing human pancreatic neuroendocrine tumour (PNET) samples from 120 patients were obtained from Helsinki Biobank. Part of the tissue cores also contained normal tissues that had been removed together with the tumours. The sections were stained immunohistochemically for RAB24 and Ki-67 as described above.\u003c/p\u003e \u003cp\u003eQuantification of RAB24 staining intensity and Ki-67 labelling index was performed using QuPath software version 5.0.1 (Bankhead, et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Whole-slide images were opened in QuPath and set to the H-DAB image type. A TMA grid was defined using the \u003cem\u003eTMA dearrayer\u003c/em\u003e function.\u003c/p\u003e \u003cp\u003eFor the analysis of RAB24 staining intensity, RAB24-positive cells were detected and classified using the \u003cem\u003epositive cell detection\u003c/em\u003e function. Tissue folds and other artifacts were manually excluded. In PNET samples, tumour tissue, pancreatic islets, and connective tissue were manually annotated in 15\u0026ndash;20 representative areas, and the \u003cem\u003etrain object classifier\u003c/em\u003e function was used to classify the remaining tissues across the entire TMA. QuPath categorized the RAB24 staining intensity on a scale from 0 to 3, where 0 indicated no staining, 1\u0026thinsp;+\u0026thinsp;weak staining, 2\u0026thinsp;+\u0026thinsp;moderate staining, and 3\u0026thinsp;+\u0026thinsp;strong staining. Each category had a separate threshold for the RAB24 staining intensity; the values for the threshold were selected manually using scores given by a pathologist as reference. RAB24 staining intensity was then quantified using the H-score, calculated as: H-score\u0026thinsp;=\u0026thinsp;0 \u0026times; (% negative cells, 0)\u0026thinsp;+\u0026thinsp;1 \u0026times; (% weakly positive cells, 1+)\u0026thinsp;+\u0026thinsp;2 \u0026times; (% moderately positive cells, 2+)\u0026thinsp;+\u0026thinsp;3 \u0026times; (% highly positive cells, 3+). The H-score data were exported using the \u003cem\u003eshow TMA measurement\u003c/em\u003e function.\u003c/p\u003e \u003cp\u003eFor the determination of Ki-67 index, whole-slide TMA images were processed as above in QuPath. A TMA grid was defined, and Ki-67-positive nuclei were detected using the \u003cem\u003epositive cell detection\u003c/em\u003e function. Nuclei located in tissue folds were manually excluded. Tumour tissue and connective tissue were manually annotated in 10\u0026ndash;20 representative areas in randomly selected cores, and an object classifier was trained to classify tissues throughout the TMA. The percentage of Ki-67 positive nuclei in tumour tissue was exported using the \u003cem\u003eshow TMA measurement\u003c/em\u003e function.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with GraphPad Prism (GraphPad) using the tests indicated in the respective figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAge-specific Rab24 levels in mouse tissues\u003c/h2\u003e \u003cp\u003eRab24 is involved in autophagy, endocytosis, cell division and other essential cellular processes (Amaya, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Militello, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Yla-Anttila, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), making its developmental regulation of particular interest. To compare Rab24 levels between organs in different age groups, we analysed samples from the brain (cerebral cortex), heart, liver, lung, kidney, spleen, skeletal muscle (\u003cem\u003etibialis anterior\u003c/em\u003e) and pancreas by immunoblotting. The tissues were obtained from mice spanning from early postnatal age to middle-aged adulthood (1-day to 9-month-old, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We utilized a total protein stain as a loading control for the quantifications. Total protein staining has been shown to be reliable for normalizing the loading in tissues with varied proteomic profiles, as it reflects the overall protein content consistently and compensates for tissue-specific differences (Bettencourt, et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Musyaju, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFirstly, we focused on comparing Rab24 levels across multiple tissues within each age group, providing insight into organ-specific Rab24 levels. For comparative analysis between the different blots, the relative levels of Rab24 in each blot were normalized to a control sample, obtained from the liver of a 1-month-old mouse, which was loaded onto each gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-h). The liver was selected as the reference tissue due to its large size, which facilitates the preparation of ample, consistent cell extract. We did not observe any significant differences in Rab24 levels based on gender (data not shown). In 1-day and 7-day-old mice, Rab24 level varied between tissues, ranging from one fourth to 2-fold of the level in the liver control sample. The highest level was observed in the heart in both age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). A relatively high level was also observed in the brain at 1 day and 7 days, and in the kidney and skeletal muscle at 7 days. In contrast, the lowest Rab24 levels were detected in the spleen and pancreas at 1 day and 7 days, and the liver and lung at 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003eIn 14-day and 1-month-old mice, Rab24 levels increased, with particularly elevated levels observed in the brain followed by the kidney, heart and skeletal muscle at 14 days, and the kidney at 1 month (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). Conversely, the lung, spleen and pancreas showed low Rab24 levels.\u003c/p\u003e \u003cp\u003eIn 3-month-old mice, Rab24 levels of the brain showed a significant increase compared with the other tissues, which was consistent in the 6 and 9-month samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h). In addition, Rab24 levels remained elevated in the kidney in the 3, 6 and 9-month samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h).\u003c/p\u003e \u003cp\u003eOverall, these findings revealed dynamic changes in Rab24 levels across different tissues during postnatal development and aging, suggesting potential tissue-specific functions for Rab24.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTissue-specific Rab24 levels according to age\u003c/h2\u003e \u003cp\u003eTo enable more reliable comparisons of Rab24 levels within each organ during postnatal development and aging, we conducted an additional set of western blots in which all samples from each tissue were loaded on the same gel. This allowed us to minimize gel-to-gel variability and to enhance the resolution of age-related changes within each tissue. In these comparisons, the Rab24 levels were normalized to the level observed in the 1-day-old sample in each tissue, because comparative analysis between the different blots was not necessary.\u003c/p\u003e \u003cp\u003eRab24 levels in the brain significantly increased starting at 14 days of age, with a 4-5-fold increase compared to the 1-day and 7-day-old samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The elevated level was sustained in the 1-month, 3-month, 6-month and 9-month-old brain, suggesting that Rab24 may play a role in supporting brain function during later stages of development and aging.\u003c/p\u003e \u003cp\u003eIn contrast to the brain, Rab24 levels in the heart and skeletal muscle peaked during early postnatal development (1\u0026ndash;14 days), followed by a significant reduction in adult tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Rab24 levels in the pancreas and liver also displayed a notable pattern according to age. In both organs, Rab24 levels were initially lower in 1-day-old tissue, rose significantly by 7 days in the pancreas and by 14 days in the liver, and then decreased at 1 month of age, and stayed at the lower level in the 3- and 9-month samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e).\u003c/p\u003e \u003cp\u003eIn contrast to the dynamic changes seen in the tissues mentioned above, Rab24 levels in the kidney remained stable across all age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Rab24 levels in the spleen and lung also showed less variability according to age (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, h).\u003c/p\u003e \u003cp\u003eCollectively, these findings highlight distinct patterns of Rab24 levels between different tissues, and according to age in each tissue, indicating the existence of tissue-specific regulatory mechanisms of Rab24 protein levels. In the brain, Rab24 levels increased after early development and remained elevated, suggesting a sustained role in postnatal housekeeping. Conversely, in the heart, muscle, pancreas, and liver, higher Rab24 level was linked to early developmental stages, decreasing as the tissues matured. Finally, the kidney, spleen, and lung exhibited more stable Rab24 levels across all tested age groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn the brain, Rab24 is predominantly localized to neurons\u003c/h2\u003e \u003cp\u003eGiven that the brain exhibited the highest levels of Rab24 among all tested organs, we next investigated Rab24 localization across different brain regions and cell types using immunohistochemistry (IHC). To explore potential changes in staining with age, we analysed brain tissue from mice ranging from 7 days to 6 months of age. Based on the morphology of the cells with a positive IHC signal, Rab24 was predominantly localized to neurons in various brain regions.\u003c/p\u003e \u003cp\u003eNotably, Purkinje cells in the cerebellum exhibited Rab24 staining across all examined age groups, from postnatal day 7 to 6-months, with minimal age-related changes in staining intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). In contrast, Rab24 staining was either absent or low in many neuronal populations of 7-day-old mice (Fig. S3). However, in older mice (1, 3, and 6 months), Rab24 levels increased in many types of neurons in different brain regions, consistent with our western blot data (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Region-specific analysis revealed Rab24-positive neurons throughout the thalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). Within the hippocampus, weak to moderate Rab24 staining was detected in neurons of the dentate gyrus, as well as in the CA1 and CA3-CA4 subfields (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, d-f). In the isocortex, Rab24 staining was observed in neurons distributed across the entire cortical mantle (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, g).\u003c/p\u003e \u003cp\u003eIn the midbrain, Rab24 was prominently stained in neurons of several sites, including the substantia nigra, red nucleus, mesencephalic trigeminal nucleus and interpeduncular nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, h-k). Similarly, in the hindbrain, Rab24 staining was high in neurons of the trigeminal motor nucleus, pontine grey, and reticular nucleus of the pons (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, l-n), and low to moderate in neurons of the cochlear nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, o).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRab24 staining in different types of epithelial cells\u003c/h2\u003e \u003cp\u003eIHC analysis revealed Rab24 staining in epithelial cells across multiple organs, with notable age-dependent differences in staining patterns. While 7-day-old mice exhibited somewhat varying epithelial staining patterns, tissues of older mice (1, 3, and 6 months) displayed more consistent staining intensities. Representative images from 7-day-old and 1-month-old mice show the age-related differences in epithelial Rab24 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-h).\u003c/p\u003e \u003cp\u003eEpendymal cells line the ventricles in the brain. In the ependymal layer, Rab24 staining was stronger compared to the surrounding brain tissue in 7-day-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). However, in older mice, Rab24 staining in the ependyma was more comparable to that of the adjacent tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn the kidney, Rab24 staining followed a similar pattern in younger and older animals, with higher staining in the epithelial cells of the proximal convoluted tubules compared to the distal tubules and glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). The less differentiated tubules showed weaker Rab24 staining in the 7-day-old kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In 1-month-old kidney, Rab24 staining was slightly stronger in the proximal tubules than in the distal tubules. Additionally, Rab24 staining was also observed in the parietal cells of the Bowman\u0026rsquo;s capsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eIn the lungs, Rab24 staining was prominent in the bronchiolar epithelium across all age groups, but differences were observed in the alveolar lining between young and old tissue. In 7-day-old mice, pneumocytes lining the alveolar walls showed stronger Rab24 staining compared to older animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f). Despite this age-related decrease, the bronchiolar epithelial cells maintained consistently high Rab24 levels throughout all age groups.\u003c/p\u003e \u003cp\u003eIn the exocrine pancreas, Rab24 staining was notably stronger in the acinar epithelial cells of 7-day-old mice compared to older animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h). The endocrine cells of the islets of Langerhans consistently showed stronger Rab24 staining compared to the surrounding exocrine tissue across all age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, h).\u003c/p\u003e \u003cp\u003eFinally, the multicancer TMAs included also normal tissues from some organs, which enabled comparisons of the Rab24 staining in mouse tissues with the corresponding human adult tissues. Analysis of the human kidney, lung and pancreas revealed RAB24 staining patterns similar to those observed in adult mouse tissues (Fig. S4a-d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRab24 staining in the heart, skeletal muscle, liver and spleen\u003c/h2\u003e \u003cp\u003eIn the heart, Rab24 staining was moderate in cardiomyocytes of 7-day-old animals with markedly reduced staining in older mice (1, 3, and 6 months, Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). In the older mice, both cardiomyocytes and cardiac endothelial cells showed low Rab24 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). A similar but less prominent age-dependent decrease in Rab24 staining was observed in the skeletal muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). In contrast, Rab24 staining in the liver remained low in all age groups included in the IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). In the spleen, Rab24 staining was moderate in both 7-day-old and older animals, with uniform staining across the white and red pulp (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). Spleen macrophages were weakly-stained in 7-day-old tissue, and moderately stained in 1-month old and older tissue.\u003c/p\u003e \u003cp\u003eAnalysis of human adult tissues from the skeletal muscle and liver revealed RAB24 staining patterns similar to those observed in the adult mouse tissues (Fig. S4e, f).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRAB24 levels in human cancers\u003c/h2\u003e \u003cp\u003eRAB24\u0026rsquo;s known involvement in autophagy (Yla-Anttila, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), endocytosis (Amaya, et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and mitosis (Militello, et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), processes frequently dysregulated in cancer, suggests that its altered expression may contribute to malignant transformation and tumour progression. Given the dynamic and tissue-specific levels of mouse Rab24 observed during postnatal development and aging, we sought to investigate whether RAB24 levels are altered in cancer. To address this, we analysed RAB24 levels by IHC in 75 tumour types originating from 220 patients using tissue microarrays (TMAs) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). RAB24 staining intensity was quantified using the H-scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-f), which were calculated with the QuPath software (Bankhead, et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The H-score is a semi-quantitative metric that combines staining intensity and the percentage of positively stained cells, providing a standardised method for comparing staining intensities in tissue samples. Frequency distribution of the H-scores revealed that RAB24 staining in the cancers included in the TMAs predominantly fall within the moderate range, with 65% of samples exhibiting H-scores between 100 and 199, while the maximum is 300 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). Strong RAB24 staining (H-score\u0026thinsp;\u0026ge;\u0026thinsp;200) was observed in 30.6% of the cancers, while low staining (H-score\u0026thinsp;\u0026le;\u0026thinsp;99) was identified in the remaining 4.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). In comparison, normal tissues removed alongside the tumours during surgery exhibited a more balanced distribution: 30.7% had lower RAB24 staining, 55.4% moderate staining, and 13.9% high staining (Fig. S5a).\u003c/p\u003e \u003cp\u003eTo provide an overview, we summarized the findings for all 75 cancer types in a table that includes their tissue of origin, average RAB24 H-scores in malignant and normal tissues, the difference of H-scores between cancer tissue and normal tissue, and the number of patients per cancer (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The data reveal distinct patterns in RAB24 levels across cancers. A majority of cancer types demonstrated elevated RAB24 levels in malignant tissues compared to normal tissues, including several breast and skin cancer subtypes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). Conversely, 24% of cancers, particularly those of the digestive system (e.g., pancreas, stomach, appendix) and urinary tract, showed reduced RAB24 levels in malignant tissues compared to normal tissues (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eh). A subset of cancers showed negligible changes in RAB24 levels (difference in H-score below 20) between malignant and normal tissues (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor further analysis, the 75 cancer types were categorized into 21 groups based on tissue or organ of origin (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. S5b). This categorization facilitated cross-comparison of RAB24 levels across diverse cancer types. For example, cancers of the thyroid, skin and pancreas displayed high RAB24 staining, while cancers of the esophagus, soft tissue and stomach showed lower staining levels (Fig. S5b). Among these cancers, only soft tissue sarcomas exhibited a statistically significant increase in RAB24 staining in malignant versus normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). Other cancers demonstrated non-significant trends of increased RAB24 staining in malignant tissue (Fig. S5c-h), higher levels in normal tissues (Fig. S5i-k), or no substantial difference between normal and cancer tissue (Fig. S5l-n).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRAB24 levels in pancreatic neuroendocrine tumours\u003c/h2\u003e \u003cp\u003eThe expression level of RAB24 was reported to be decreased in patient samples and cell lines of pancreatic adenocarcinoma, which originates from the exocrine cells. RAB24 was also observed to be an independent low-risk factor in this cancer type (Deng, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yu, et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings motivated us to investigate RAB24 protein levels in pancreatic neuroendocrine tumours (PNETs), which originate from the endocrine pancreatic islet cells.\u003c/p\u003e \u003cp\u003eWe analysed RAB24 protein levels using TMAs containing samples from 120 patients with PNET. The TMAs contained tissue samples from three regions: the tumour, the tumour edge, and normal pancreatic tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c). IHC staining for RAB24 was performed, and the H-scores were quantified using QuPath software. In tumour and tumour edge samples, the H-score was calculated separately for tumour cells and connective tissue, while in normal tissue samples, the H-score was determined for islet cells (the tissue of origin for PNETs) and connective tissue.\u003c/p\u003e \u003cp\u003eThe analysis revealed significantly higher RAB24 staining in pancreatic islet cells from normal tissue compared to the tumour and tumour edge samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). No difference in RAB24 levels was observed between tumour and tumour edge samples. Furthermore, in connective tissue, RAB24 staining was significantly lower than in tumour cells. In normal tissue, RAB24 staining was lower in connective tissue compared to islet cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Notably, no differences in RAB24 levels were detected between connective tissue in tumour, tumour edge, and normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo determine whether RAB24 protein levels vary with tumour grade, we performed IHC staining for Ki-67, a well-established proliferation marker (Fig. S6). Based on Ki-67 indices (Nagtegaal, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the majority of PNET samples (92.2%) were classified as low-grade (G1, 0\u0026ndash;3% Ki-67-positive cells), while 6% were classified as intermediate-grade (G2, 3\u0026ndash;20% Ki-67-positive cells), and 1.7% as high-grade (G3, more than 20% Ki-67-positive cells). Quantitative analysis of RAB24 H-scores revealed a significant decrease in RAB24 expression in higher-grade tumours, with PNET G1 samples displaying higher RAB24 levels compared to G2 and G3 tumours (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e8\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study provides a comprehensive analysis of Rab24 levels in mouse tissues from early postnatal age to adulthood, revealing distinct tissue-specific patterns and dynamic changes with age. We also analysed RAB24 levels in human cancers in comparison to peritumoral tissues, and found disease-specific alterations in Rab24 levels in the tumours.\u003c/p\u003e\n\u003cp\u003eAge- and tissue-specific changes in Rab24 level in mouse tissues\u003c/p\u003e\n\u003cp\u003eRab24 levels in several tissues underwent dynamic changes during early postnatal development, followed by stabilization in adulthood. These findings indicate that Rab24 may contribute to cellular processes during organ development, with a potential role in tissue homeostasis in maturity. While our data do not provide direct evidence that Rab24 is critical for these processes, previous studies have demonstrated its essential role in Purkinje neuron survival (Agler, et al., 2014).\u003c/p\u003e\n\u003cp\u003eIn the brain, Rab24 levels increased significantly between 7 and 14 days of age and remained elevated into adulthood. Immunohistochemistry showed that different types of neurons have the highest Rab24 levels. The significant increase in brain Rab24 level coincides with the peak of synaptogenesis, a period marked by heightened neuronal activity and metabolic demands\u0026nbsp;(Faria-Pereira and Morais, 2022, Li, et al., 2010).\u0026nbsp;Interestingly, Rab24 has been found to localize to synaptic vesicles\u0026nbsp;(Taoufiq, et al., 2020). These findings suggest a possible role for Rab24 in neuronal differentiation and long-term neuronal maintenance. In addition to synaptogenesis, also other processes are activated in the mouse brain during postnatal days 1-10, including production, migration and differentiation of additional neurons, and selective elimination of unnecessary neurons by programmed cell death\u0026nbsp;(Chen, et al., 2017a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWithin the brain, immunohistochemistry confirmed robust Rab24 levels in different types of neurons, including neurons involved in sensory processing, motor control and coordination, such as Purkinje cells. Interestingly, Rab24 protein staining in Purkinje cells was already evident at 7 days of age, and stayed similar until adulthood. This aligns with prior evidence showing that a Rab24 mutation leads to Purkinje cell degeneration in dogs\u0026nbsp;(Agler, et al., 2014),\u0026nbsp;underscoring the essential role of Rab24 in maintaining this specialized neuronal population.\u003c/p\u003e\n\u003cp\u003eIn several peripheral tissues, including the heart, skeletal muscle, pancreas and liver, Rab24 levels decreased after early postnatal development, i.e., after 14 days of age. The elevated Rab24 levels coincide with a time of increased biosynthetic and metabolic activity associated with organ maturation. In the heart and skeletal muscle, Rab24 levels peaked between 1 and 14 days of age, a period of rapid cellular growth and differentiation (Gattazzo, et al., 2020, Li, et al., 1996), before decreasing to stable levels in adulthood. The initially higher levels of Rab24 in the heart coincide with a period of rapid growth and cellular differentiation of cardiomyocytes (Piquereau, et al., 2010). Similarly, Rab24 level in the liver and pancreas was notably elevated at 7 and 14 days (pancreas) or 14 days (liver) of age compared to older tissues, correlating with the high metabolic and biosynthetic demands of these tissues during postnatal development (Bonner-Weir, et al., 2016, Liang, et al., 2022). Notably, Rab24 level in the pancreatic islets of Langerhans remained consistently higher than in the surrounding exocrine tissue across all ages, suggesting a sustained role for Rab24 in the hormone-secreting cells.\u003c/p\u003e\n\u003cp\u003eIn the lung, Rab24 staining was elevated in both the alveolar lining cells and the bronchiolar epithelium of 7-day-old mice. The alveolar lining appeared denser in the young animals, with stronger Rab24 staining compared to older mice. Active alveolar remodelling and expansion during early postnatal development coincide with the 7-day sample collection \u0026nbsp; (Negretti, et al., 2021). In contrast, Rab24 staining in the club cells of the bronchiolar epithelium remained consistently high across all ages. These cells synthesise and secrete the lining fluid of the respiratory epithelium (Blackburn, et al., 2023).\u003c/p\u003e\n\u003cp\u003eOrgans such as the kidney and spleen displayed relatively stable Rab24 levels across all age groups, suggesting a more consistent demand in Rab24 protein. In the kidney, Rab24 was uniformly stained in the proximal convoluted tubular epithelial cells. Greater variability in Rab24 staining was observed in the kidney tubules of 7-day-old mice. Stronger staining was observed particularly in larger, more differentiated tubules, suggesting a role for Rab24 during tubular differentiation or function. Rab24 was also stained in the parietal epithelial cells of the Bowman’s capsule but not in podocytes of the glomerulus. In the spleen, Rab24 was stained in both the white and the red pulp. Within the red pulp of 1-month-old and older animals, Rab24 was enriched in large cells, likely macrophages, which are critical for immune surveillance and phagocytosis.\u003c/p\u003e\n\u003cp\u003eThe differences in Rab24 levels between 7 and 14-day-old and older animals suggest that Rab24 is developmentally regulated. Because pathways such as autophagy and endocytosis, where Rab24 has documented roles (Amaya, et al., 2016, Yla-Anttila, et al., 2015), are essential for both differentiation and maintenance, Rab24 may contribute to these processes throughout life rather than having distinct functions at different stages. During early postnatal development, Rab24 may contribute to neuronal differentiation and/or maintenance, epithelial remodelling, and organ maturation. In adulthood, the role of Rab24 may shift towards maintenance of homeostasis by regulating similar mechanisms.\u003c/p\u003e\n\u003cp\u003eRab24 staining in epithelial cells suggests a potential role in the biology of this cell type. Epithelial cells undergo continuous turnover and require tightly regulated intracellular trafficking processes, including endocytosis and autophagy, to maintain tissue integrity (Schwarz, et al., 2007, Wong, et al., 2019). Many epithelial cells have also active secretory functions: ependymal cells secrete the cerebrospinal fluid, and pancreatic islet cells secrete hormones. Epithelial cells can also be active in endocytosis, like the kidney proximal tubule epithelium. In developing tissues, Rab24 could contribute to epithelial remodelling and differentiation, while in mature tissues, it may support homeostatic processes such as barrier maintenance and cellular renewal. Further studies are needed to define the precise contributions of Rab24 to epithelial cell functions as well as age and tissue-specific roles.\u003c/p\u003e\n\u003cp\u003eInsights from the mouse data highlight the developmental regulation of Rab24, with levels peaking during critical periods of organ development in the brain, heart, muscle, pancreas and liver. The age-dependent Rab24 levels suggest that Rab24 levels may also vary during different stages of cancer progression, similar to the surge observed in early postnatal development.\u003c/p\u003e\n\u003cp\u003eRab24 levels in cancers\u003c/p\u003e\n\u003cp\u003eThe dynamic changes in Rab24 levels observed during postnatal development in mice, particularly surges during periods of organ maturation, may mirror mechanisms exploited or disrupted during tumorigenesis. For instance, the processes governing Rab24’s involvement in endocytosis, autophagy, as well as mitosis, cell migration and invasion could be exploited by cancer cells to support their growth, survival, and metastasis (Amaya, et al., 2016, Chen, et al., 2017b, Militello, et al., 2013, Yla-Anttila, et al., 2015). Furthermore, the tissue-specific dynamic variations in Rab24 levels with age emphasize the importance of context in the possible roles of Rab24. Dysregulation in cancer may arise from perturbations in the finely-tuned developmental programs.\u003c/p\u003e\n\u003cp\u003eOur results showed that RAB24 levels in cancers of the breast and skin were higher than in the corresponding normal tissues, while the opposite was found in cancers of the digestive system and the urinary tract. The observed heterogeneity in RAB24 levels across various cancer types aligns with its multifaceted roles in cellular processes, which may be hijacked or altered in cancer cells to support oncogenic processes. For example, elevated RAB24 levels in HCC and HCC cell lines have been associated with enhanced growth and metastasis (He, et al., 2002, Yang, et al., 2020, Zhang, et al., 2020, Zhu, et al., 2020). This elevation reflects the role of RAB24 in promoting malignant phenotypes such as epithelial-mesenchymal transition (EMT), vasculogenic mimicry, and cell adhesion (Chen, et al., 2017b). RAB24 is also an unfavourable prognostic marker in prostate cancer (Hu, et al., 2020). Conversely, reduced RAB24 expression in other cancers may indicate downregulation in favour of alternative survival mechanisms, reflecting the adaptability of cancer cells in diverse tissue environments.\u003c/p\u003e\n\u003cp\u003eOur findings revealed that RAB24 levels are significantly reduced in pancreatic neuroendocrine tumours (PNETs) compared to normal pancreatic islet cells. Given the known involvement of RAB24 in autophagy and endocytosis, the downregulation in PNETs may reflect alterations in pathways that contribute to tumour progression. Notably, while RAB24 staining was consistently lower in tumour cells than in normal islet cells, H-scores in malignant tissues exhibited substantial variability among patients, with values varying between 294.1 and 129.6. To investigate whether this variability correlated with tumour grades, we assessed the tumour grades using Ki-67 staining. Our analysis revealed an inverse correlation between RAB24 level and tumour grade, further supporting a potential role of RAB24 in PNET biology. PNETs classified as G1 exhibited significantly higher RAB24 levels than those classified as G2 or G3. Although the number of G2 and G3 samples in our cohort was limited, our findings suggest that RAB24 downregulation may be linked to more aggressive tumour phenotypes. Given that Ki-67 is a well-established marker of cell proliferation, the inverse relationship between Ki-67 and RAB24 staining suggests that RAB24 downregulation may be associated with increased proliferative capacity and tumour aggressiveness in PNETs. Future studies with larger patient cohorts should explore whether RAB24 levels correlate with clinical parameters such as patient survival or response to therapy, and whether loss of RAB24 contributes functionally to increased malignancy.\u003c/p\u003e\n\u003cp\u003eCollectively, our findings provide a comprehensive overview of RAB24 levels across a wide spectrum of human cancers. The observed differences in RAB24 levels between malignant and normal tissues, as well as among cancer types, suggest that RAB24 may play context-dependent roles in malignancy. These insights lay the groundwork for future studies to elucidate RAB24’s functional contributions to tumorigenesis and its potential as a diagnostic tool or therapeutic target in cancer.\u003c/p\u003e"},{"header":"Conclusions and implications","content":"\u003cp\u003eIn this study, tissue and age-specific levels of Rab24 in mouse were observed by western blot and confirmed in-situ by IHC. The dynamic changes in Rab24 levels during early development suggest roles in organ maturation in certain tissues including the brain, heart, skeletal muscle, pancreas and liver while the stable levels in adulthood point to maintenance functions. Further, our results showed that RAB24 levels in cancers of the breast and skin were higher than in the corresponding normal tissues, while the opposite was found in cancers of the digestive system and the urinary tract. In pancreatic PNET, RAB24 levels were lower than in normal islet cells.\u003c/p\u003e\n\u003cp\u003eFuture research is needed on possible contributions of Rab24 to age-related disorders and on its potential as a biomarker or therapeutic target in cancer and other diseases. Further studies of the molecular mechanisms of Rab24 in health and disease will be pivotal in harnessing its diagnostic and therapeutic potential.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank the core facilities at the Institute of Biomedicine, University of Turku: Histology core facility for assisting with the sectioning of paraffin-embedded mouse tissues, and Medisiina Imaging Center for availability of the slide scanner. Hira Javed is also thanked for assistance in paraffin sectioning. We thank\u0026nbsp;Kati Kuipers and\u0026nbsp;the Finnish Center for Laboratory Animal Pathology, University of Helsinki,\u0026nbsp;for the Ki-67 staining of the PNET sections.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis project was funded by Marie Skłodowska-Curie ETN grant under the European Union’s Horizon 2020 Research and Innovation Programme (Grant Agreement No 765912, DRIVE), the Institute of Biomedicine, University of Turku, Magnus Ehrnrooth Foundation (March 6, 2021), and the Research Council of Finland (grant No 351215). H.G.M.R was supported by the Finnish Cultural Foundation Central Fund (grant No 00220857) and the Turku University Foundation (grant No 081769). P.S. was supported by\u0026nbsp;RESET (Resilient and Just Systems) research profiling funding from the Research Council of Finland (PROFI7 2023–2028).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgler C, Nielsen DM, Urkasemsin G, Singleton A, Tonomura N, Sigurdsson S, Tang R, Linder K, Arepalli S, Hernandez D, Lindblad-Toh K, van de Leemput J, Motsinger-Reif A, O'Brien DP, Bell J, Harris T, Steinberg S, Olby NJ (2014) Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24. Plos Genet 10:e1003991\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaya C, Militello RD, Calligaris SD, Colombo MI (2016) Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation. 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Aging 12:14582\u0026ndash;14592\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rab24, Mouse tissues, Cancer, Pancreatic neuroendocrine tumours ","lastPublishedDoi":"10.21203/rs.3.rs-6176716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6176716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRab24 is an unusual member of the Rab family of small GTPases, implicated in autophagy, endocytosis and cell division. In order to elucidate possible organ and age-specific roles of Rab24, we investigated tissue-specific levels of Rab24 in mice by western blotting and immuno­histo­chemistry in samples from postnatal day one to 9 months of age. In adult mice, the highest protein levels were found in the brain followed by the kidney, while Rab24 levels in the pancreas, spleen, liver, lung, heart, and skeletal muscle were lower. Dynamic changes in Rab24 levels were observed during early postnatal development, with a sharp increase in the brain at postnatal day 14, after which the level remained high into adulthood. In the heart, skeletal muscle, pancreas and liver, higher Rab24 levels were observed during the first two postnatal weeks, after which the levels dropped and stayed low until adulthood. The age-dependent changes suggest organ-specific roles for Rab24 in development and maturation. Immunohistochemistry of the brain revealed that Rab24 was mostly present in neuronal cells. Also, epithelial cells in several tissues showed high Rab24 levels. These results suggest roles for Rab24 in neuronal and epithelial maintenance. Furthermore, we also analysed immunohistochemical staining for RAB24 in human cancers and normal tissues. RAB24 staining in cancers of the breast and skin was higher than in the corresponding normal tissues, while it was reduced in cancers of the digestive system and the urinary tract. In pancreatic neuroendocrine tumours that originate from islet cells, RAB24 levels were lower than in normal pancreatic islet cells. Collectively, our findings provide a comprehensive overview of RAB24 levels across a wide spectrum of human cancers. The observed differences in RAB24 levels between cancer types and between malignant and normal tissues, suggest that RAB24 may play context-dependent roles in malignancy.\u003c/p\u003e","manuscriptTitle":"Rab24 protein levels show dynamic changes in mouse tissues and human cancers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 04:02:28","doi":"10.21203/rs.3.rs-6176716/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T14:03:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-04T12:24:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-02T09:54:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219036882025203684175932239381177263467","date":"2025-03-24T00:32:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337365392337082030461773055585206115424","date":"2025-03-20T00:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186556652763550400732103664167161907573","date":"2025-03-16T06:14:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-13T17:50:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-11T08:28:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-11T08:24:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2025-03-07T08:52:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bc3ba16b-d8b0-4166-aec7-9004365c8882","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-27T21:09:41+00:00","versionOfRecord":{"articleIdentity":"rs-6176716","link":"https://doi.org/10.1007/s00441-025-04043-4","journal":{"identity":"cell-and-tissue-research","isVorOnly":false,"title":"Cell and Tissue Research"},"publishedOn":"2026-01-27 00:00:00","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-03-24 04:02:28","video":"","vorDoi":"10.1007/s00441-025-04043-4","vorDoiUrl":"https://doi.org/10.1007/s00441-025-04043-4","workflowStages":[]},"version":"v1","identity":"rs-6176716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6176716","identity":"rs-6176716","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}