Spatial Patterns and Prognostic Relevance of CD1a Immature and CD208 Mature Dendritic Cells in Colorectal Cancer From Tumour-adjacent Mucosa to Liver Metastases

Spatial Patterns and Prognostic Relevance of CD1a Immature and CD208 Mature Dendritic Cells in Colorectal Cancer From Tumour-adjacent Mucosa to Liver Metastases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Spatial Patterns and Prognostic Relevance of CD1a Immature and CD208 Mature Dendritic Cells in Colorectal Cancer From Tumour-adjacent Mucosa to Liver Metastases Sergii Pavlov, Esraa Ali, Wenjing Ye, Lenka Červenková, Filip Ambrozkiewicz, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7588685/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 11 You are reading this latest preprint version Abstract Background . The prognostic role of dendritic cells (DCs) in colorectal cancer (CRC) and paired liver metastases (LM) remains unclear, particularly regarding the dynamics of immature CD1a⁺ and mature CD208⁺ subsets across anatomical compartments and synchronous versus metachronous disease. Patients and methods . This retrospective cohort included patients undergoing resection of primary CRC (pCRC) and synchronous LM (N = 55) or metachronous LM (N = 44). Immunohistochemical staining for CD1a and CD208 was performed on non tumour adjacent normal mucosa (NAM), tumour center (TC), inner and outer invasive margins (IM, OM), and peritumoural tissue (PT). Cell densities were quantified on whole-slide images using QuPath software and correlated with overall survival (OS). Results . CD1a⁺ DCs were nearly absent in NAM but enriched in pCRC TC, whereas CD208⁺ DCs predominated in NAM lymphoid aggregates and peripheral compartments of pCRC and LM. CD1a⁺ cells followed a TC/IM > OM/PT gradient, while CD208⁺ cells showed the opposite, consistent with a recruitment–maturation axis. In synchronous cases, CD1a⁺ densities were higher in pCRC than LM, supporting the role of the primary tumour as a “monocyte reservoir.” Survival analysis revealed that high CD208⁺ density in TC of synchronous LM (HR = 0.47; p = 0.033) and high CD1a⁺ density in TC of metachronous LM (HR = 0.44; p = 0.050) were both associated with reduced mortality risk. Conclusion . This study provides the first detailed mapping of CD1a⁺ and CD208⁺ DCs across NAM, pCRC and paired LM, indicating that their prognostic impact is determined not only by absolute numbers but, more importantly, by compartmental distribution and the timing of metastasis. colorectal cancer synchronous and metachronous liver metastases dendritic cells CD1a CD208 spatial immune profiling overall survival Figures Figure 1 Figure 2 Figure 3 Introduction Colorectal cancer (CRC) is a leading cause of cancer-related deaths. Despite improved treatment, up to 60% of patients ultimately develop distant metastases, the liver being the main site because of portal drainage and a permissive pre‑metastatic niche ( 1 ). The immune contexture of tumour microenvironment (TME) in both primary and metastatic tumours is now recognized as a key determinant of whether disseminated CRC cells are eliminated, held in dormancy, or allowed to progress ( 2 – 4 ). Among the diverse immune populations shaping the CRC microenvironment, dendritic cells (DCs) represent central orchestrators of antitumour immunity ( 5 ). DCs are a highly heterogeneous cell population comprised of several subsets with distinct origins, locations, migratory and functional properties ( 4 ), which bridge innate and adaptive immune responses ( 6 ). First subset of classical DCs (cDC1) are the principal cells for cross-presenting tumour antigens to CD8⁺ T lymphocytes and initiating cytotoxic responses, besides priming CD4⁺ T cells ( 7 ). The cDC2 subset activates CD4⁺ T lymphocytes and drives their polarization into diverse T-helper populations ( 8 , 9 ). The contribution of cDC1 and cDC2 to antitumour immunity is context-dependent and often contradictory, complicating evaluation of their prognostic impact in CRC ( 9 ). Plasmacytoid DCs (pDCs) are characterized by robust production of type I interferons and, under physiological conditions, play a key role in antiviral defense ( 10 ). It is known that pDCs can infiltrate solid tumours, although they are relatively scarce ( 11 , 12 ) and their antitumour efficacy is limited ( 11 , 13 ). Monocytic DCs (moDCs) arise from circulating monocytes during precancerous transformation and are dominant in tumour tissue ( 14 ). Although moDs can activate CD4⁺ and CD8⁺ T cells and cross-present antigens, dendritic-cell maturation in tumour tissue is frequently dysregulated, shifting the balance toward immature states ( 15 , 16 ). At present, CD1a is commonly accepted as a marker of monocytic ( 17 , 18 ), and immature DCs ( 19 , 20 ), whereas CD208 (DC-LAMP) is recognized as a marker of mature DCs ( 21 , 22 ). However, the specific roles of DCs subsets and their prognostic significance in CRC progression remain only partially understood and are mostly contradictory. Several studies have reported a positive prognostic impact of CD1a + DCs in CRC ( 19 , 23 ), while other reports have described the immunosuppressive role of CD1a + DCs and their negative influence on patient survival in CRC ( 19 , 24 , 25 ). Similarly, CD208 + DCs have been associated with a favorable prognosis in CRC in ( 21 , 26 ), whereas other studies have linked it to adverse outcomes ( 15 ). In the gut mucosa, immature DCs constantly sample the environment by taking up antigens from the lumen through phagocytosis and macropinocytosis. ( 27 ). Upon maturation and migration to gut-associated lymphoid tissues, these DCs present processed antigens to T cells, which can then elicit the adaptive immune response towards pathogen clearance or tolerogenic pathways for maintaining homeostasis ( 28 ). However, evidence remains limited on how DCs in non tumor adjacent mucosa (NAM) may influence the initiation, progression and clinical course of colorectal cancer. It remains unclear whether the spatial localization of DCs has prognostic significance in CRC. High-resolution mapping by Miller et al. revealed a sharp dichotomy between tumour center and invasive margin, with DCs density peaking in invasive margin, where high cell densities predict better survival ( 29 ). Comparative studies assessing DCs distribution and prognostic impact between primary CRC and liver metastases, critical for understanding the loss of immune surveillance in metastases ( 5 ), are lacking. A synthesis of literature has highlighted three major gaps: limited data on quantitative and spatial profiles of CD1a⁺ and CD208⁺ DCs in CRC from NAM through primary tumour to liver metastases; absence of studies addressing landscape of DCs in synchronous versus metachronous CRC; few, often inconsistent, cohort studies linking these subsets to patient outcomes. The aim of the present study was therefore to map the distribution of CD1a⁺ and CD208⁺ DCs from non-tumour mucosa through pCRC to LM, and to determine how compartment-specific cell counts associate with overall survival in synchronous versus metachronous cases. Material and methods Patients All patients who underwent curative resection of pCRC followed by hepatic resection for the first recurrence of CRC at Pilsen University Hospital between 1999 and 2021 were retrospectively reviewed. LM detected at pCRC diagnosis defined the stage IV synchronous cohort (n = 80); LM identified 17 (1–59) months (median (interval) after pCRC resection defined the stage I–III metachronous cohort (n = 100). Inclusion criteria required first-episode LM, curative-intent surgery for both pCRC and LM, complete clinical and survival data, and available good quality formalin fixed-paraffin embedded (FFPE) tissue. Exclusion criteria were multiple primaries, pre-operative extra-hepatic disease, previous liver resections, neoadjuvant chemoradiotherapy, emergency surgery, or death within 30 days post-operation. Ninety-nine patients fulfilled all criteria (55 with synchronous LM and 44 with metachronous LM). Demographic, pathological and clinical variables were extracted (Table S1 ). Tumours were staged according to the criteria of AJCC 8th edition; most were histological type NOS, grade 2. The cohorts differed only in median LM size (larger in the metachronous group) and in the proportion of patients receiving adjuvant FOLFOX (lower in the metachronous group). The study followed the Declaration of Helsinki (2013) and was approved by the local ethics committee (300/2020, 17 June 2020). Pathology and immunohistology FFPE tissues of pCRC, LM and NAM from each patient were identified and cut into 4-µm sections. Sample for NAM was taken from the resection margin (oral or aboral), which was the closest to the tumour, with a median distance from the tumour 34 mm (range: 4-200 mm). In case of multiple LMs, we selected the metastatic tumour with the least regressive changes. One or two tissue sections were mounted onto BOND Plus Microscope Slides (Cat#00270, Leica Biosystems Newcastle Ltd., Newcastle, UK). Immunohistochemical detection of CD1a and CD208 cells was performed using fully automated BOND RXm IHC/ISH stainer. Ready-to-use monoclonal primary antibodies for CD1a (clone MTB1) from Leica Biosystems (Newcastle Ltd., United Kingdom), CD208 (clone EPR24265-8) from Abcam (Abcam Ltd., United Kingdom) were used. Binding of primary antibodies with their targets was visualized using horseradish peroxidase (HRP)-linker antibody conjugate system (Bond™ Polymer Refine Detection). Sections were counterstained with Mayer's haematoxylin and embedded into Micromount mounting medium (Leica Biosystems Newcastle Ltd., United Kingdom). Appropriate positive (tonsils) and negative tissue control samples were used throughout. Image analysis Whole-slide scans were acquired using an Olympus VS200 scanner (Olympus, Shinjuku, Japan). Regions of interest (ROIs) in pCRC and LM, including tumour center (TC), inner margin (IM), outer margin (OM), and peritumour zone (PT), were annotated and analysed in QuPath v.0.4.3 using custom scripts ( https://github.com/sergii01-cuni/script_zones ). NAM was annotated as a single region above the muscularis mucosa, encompassing surface epithelium, intestinal crypts, and lamina propria, with exclusion of dysplasia, crypt lumina, and artefacts. Tumour borders were defined at the interface between malignant cell nests and adjacent non-tumour tissue, excluding luminal surface, large vessels, normal mucosa, dysplastic epithelium, muscularis propria, supportive stroma > 2 mm, extracellular mucin, fat, necrosis, abscesses, haemorrhages, and artefacts. CD1a⁺ and CD208⁺ cell densities were calculated for NAM and each ROI as the number of immunopositive cell profiles per total ROI area. To eliminate skewness in the distribution, the raw data were converted into percentile values for survival analysis and then categorized into two groups: low (below the 25th percentile) and high (25th–100th percentile). Follow-up Follow-up continued through December 2023. Median surveillance after liver metastasectomy was 84 months (95% CI 5–163) in the synchronous cohort and 61 months (95% CI 54–68) in the metachronous cohort. Patients were reviewed every three months for two years, then semi-annually; each visit included tumour-marker assays, chest X-ray, abdominal ultrasound, and CT. PET or MRI was added at the multidisciplinary team’s discretion. Outcomes The endpoint of the study was overall survival (OS), measured from resection of LM to death from any cause. Patients without death were censored at their last follow-up. OS after LM surgery were not statistically different between groups (data not shown). Statistical methods Continuous variables with non-normal distributions were presented as median (range) and compared using the Mann–Whitney U test or, for repeated measures, Friedman ANOVA with Wilcoxon matched-pairs tests (Bonferroni adjusted). Categorical variables were expressed as counts (%). OS was estimated by Kaplan–Meier analysis and compared with the log-rank test; prognostic value of predictors was assessed by Cox regression with hazard ratios (HRs) for high vs. low groups. Multiple regression with backward stepwise elimination identified predictors of DCs density in ROIs; model quality was assessed by R² and Fisher’s F test, with elasticity coefficients quantifying covariate influence. Associations between variables were tested by Spearman correlation. Analyses used GraphPad Prism 9.0; survival modelling employed the finalfit package, with significant Cox results visualised in survival and survminer. Two-sided p < 0.05 was considered significant. Results 1. Morphology and topography of CD1a⁺ and CD208⁺ DCs in NAM, pCRC and LM Rounded CD1a⁺ DCs were extremely rare in the lamina propria of NAM; in addition, they were occasionally present within the marginal zone of lymphoid follicles (LF) associated with NAM (Figure S1 a). Rounded and stellate-shaped CD208⁺ DCs were larger, they spanned the lamina propria forming micro-clusters of 10–20 cells (Fig. 1Sb) and also accumulated in mantle and marginal zones of LF (Figure S1 c). In pCRC, CD1a⁺ cells were sparse, confined to stromal spaces between glands (Figure S2 a) with slightly higher density at the invasive front and sporadically dispersed in PT and OM. CD208⁺ cells were abundant in the stromal regions surrounding the tumour and within lymphoid aggregates (LA) (Figure S2 b), which were more frequent toward the PT region. In LM, CD1a⁺ DCs were exceptionally scarce, appearing as solitary elements in stroma and LA (Fig. 3Sa). CD208⁺ DCs were abundant, forming micro-clusters in the TC and IM, accumulating predominantly in the LA, which were, however, more numerous in the OM (Fig. 3Sb). TC and IM of LM were also enriched in LA (Fig. 4Sa-d), which were more frequent than in pCRC, lying mainly at the tumour–liver interface. Collectively, LM thus display a pronounced imbalance between scant and dispersed immature CD1a⁺ cells and spatially organized clusters of mature CD208⁺ DCs. 2. Distribution of CD1a + and CD208 + DCs between TC of pCRC and NAM Analysis of CD1a⁺ cell distribution in NAM and in the TC of pCRC showed significantly greater cell densities in the TC of pCRC in both cohorts (Fig. 1a, b). By contrast, the density of CD208⁺ DCs was not statistically significant different between the two tissues in either group (Fig. 1a, b). The number of CD208⁺ cells within NAM was significantly higher than the number of CD1a cells in both patient groups (p = 0.0001). No statistically significant difference in densities of either CD1a⁺ or CD208⁺ cells in NAM were observed between groups. We found significant correlation between densities of CD1a + and CD208 + DC in the TC, and between CD1a + DC in the TC and NAM in the cohort with synchronous metastasis (Table S2 ), whereas in patients with metachronous metastasis densities of CD1a + DC and CD208 + DC correlated only in the TC (Table S3). 3. Distribution of CD1a and CD208 cells in pCRC and LM In pCRC, CD1a⁺ density was highest at the IM, following the order IM > TC > OM > PT in synchronous cases and IM ≈ TC > OM > PT in metachronous cases (Fig. 2 a). In LM, both cohorts showed order IM > TC > OM > PT (Fig. 2 b), with greater density in OM of synchronous LM than metachronous LM (p PT = TC ≥ IM order in both cohorts, with no intergroup differences; in LM the pattern was OM > PT > TC = IM, but IM and PT of metachronous LM contained more CD208⁺ cells than synchronous LM (both p < 0.05) (Fig. 2 c, d). CD208⁺ cells predominated over CD1a⁺ in OM and PT of both pCRC and LM in both cohorts (p < 0.05), whereas in synchronous disease CD1a⁺ cells exceeded CD208⁺ in pCRC IM (p < 0.01) and LM TC (p < 0.05). Compared between pCRC and LM, CD1a⁺ density was higher in every pCRC compartment than in the corresponding compartment of LM in the synchronous cohort (p < 0.05), while CD208⁺ density was greater in TC of pCRC in both cohorts (p < 0.05). Spearmanʼs analysis revealed significant correlations for CD1a⁺ and CD208⁺ DCs between all ROIs within pCRC and within LM (Table S3, S4), with the strongest links consistently observed between adjacent ROIs (TC and IM, IM and OM, OM and PT). In synchronous pCRC, the strongest correlations involved CD1a TC with CD208 IM and TC, whereas in LM CD1a TC correlated with CD208 OM. In metachronous pCRC, CD1a IM correlated with CD208 PT, IM, and OM, whereas in LM the key associations were CD1a TC with CD208 OM and TC. No significant correlations were found between corresponding ROIs of pCRC and LM, except for CD1a in OM (p = 0.05) in metachronous cases. Survival analysis Survival analysis demonstrated that higher CD208⁺ cell density in the TC of synchronous LM was significantly associated with reduced mortality risk (HR = 0.47; 95% CI: 0.23–0.94; p = 0.033). In contrast, increased CD1a⁺ density in the TC of metachronous LM was associated with improved survival (HR = 0.44; 95% CI: 0.19–1.00; p = 0.050), although the association was borderline. Kaplan–Meier analysis confirmed association of CD208⁺ cells in the TC of synchronous LM with longer OS (Figure S5a) with the curve showing consistently higher survival across the entire observation period. CD1a⁺ cell density in the TC of metachronous LM were significantly associated with a longer OS (Figure S5b). Based on survival analysis results, multiple linear regression was used to identify independent predictors of CD208⁺ DCs density in TC of synchronous LM and CD1a⁺ DC density in TC of metachronous LM. No valid model was obtained for CD1a, whereas in synchronous LM a robust model identified CD208 in the IM of LM as the strongest predictor (Model 1). CD208 TC of LM =1,753+0,475×CD208 IM of LM ( Model 1 ) R 2 =0.533, p<0.0001 The model was statistically significant; an increase of 10 cells in CD208 in the IM of LM corresponds to a rise of roughly 5 CD208+ cells in the TC of LM. Model stability was supported by a high R² and a significant F-test p-value. Accordingly, we constructed a model predicting density of CD208+ DC in IM of synchronous LM: CD208 IM of LM = 1.696 × CD1a TC of LM − 0.180 × CD1a IM of LM + 2.680 × CD1a PT of LM ( Model 2 ) R² = 0.679, p < 0.001 The model 2 was statistically significant and demonstrated high quality. CD1a in the TC and PT region of LM had a positive effect on density of CD208 in the IM of LM whereas CD1a in the IM of LM had a negative effect. To quantify the relative impact of each variable, elasticity coefficients were calculated (Table S5). As shown in Table 1, density of CD1a+ DCs in the TC of LM had the strongest effect on density of CD208+ DCs in the IM of LM. Associations of clinical and pathology variables with CD1a and CD208 DCs Tables S6–S8 summarize associations between clinicopathological variables and CD1a, CD208 DC densities across NAM, pCRC and LM, DCs and chemotherapy administered before and after liver resection, and DCs and FOLFOX-based chemotherapy, respectively. Statistically significant differences are indicated in the Table S6-8 legends. Discussion The immune landscape of pCRC and its metastases constitutes a dynamic system in which DCs are one the main actors of the immune response. Most previous studies have investigated DCs primarily in pCRC, often as a pooled population or restricted to the tumour core, without considering their maturation state or compartment-specific localization; in contrast, studies focusing on liver metastases are relatively few and provide only limited insight into DC heterogeneity (15,30–32), To our knowledge, this is the first study to comprehensively characterize CD1a⁺ and CD208⁺ DCs distribution across NAM and several anatomical compartments in both primary CRC and paired synchronous or metachronous LM, and to directly relate these spatial patterns to patient survival. Distribution of CD1a and CD208 DCs in NAM Previous studies have provided very limited information on the landscape of immature and mature DCs in NAM, as most investigations have concentrated on tumour tissue itself. However, establishing a baseline profile of DCs subsets in NAM could serve as a reference for assessing tumour-associated alterations. We observed high densities of CD208⁺ DCs with an almost complete absence of CD1a⁺ DCs within NAM, yielding a CD208/CD1a ratio of approximately 25:1. Mature CD208⁺ DCs in NAM were predominantly located within mucosa-associated LF, where DCs complete functional maturation and participate in antigen presentation (33) . Absence of correlation between CD208⁺ DCs in NAM and CD1a+ or CD208+ cells across different ROIs of pCRC suggest their autonomous resident profile in NAM, which is supported by the literature (27). Distribution of CD1a and CD208 DCs in pCRC and LM Most earlier studies have investigated DCs either as a pooled population or with an emphasis on the tumour core of primary or metastatic tumour, while comparative analyses between primary tumours and paired liver metastases, including fine regional assessment, remain scarce. Greater densities of CD1a+ DCs in TC of pCRC vs NAM can reflect CCL2-dependent influx of circulating monocytes that differentiate into moDCs within the TME (27,34), supplying therefore precursors for peritumoural maturation. Similar mechanism can be responsible for higher densities of CD1a+ DCs in the tumour interior of LM vs tumour exterior. Across pCRC and LM tissue in both synchronous and metachronous cohort of patients, CD1a⁺ density decreased from the TC/IM towards the OM/PT, whereas CD208⁺ density followed the opposite gradient. This consistent trend aligns with mechanisms described by Jie Chen, Yuhang Duan et al. (2023) (35), whereby hypoxic stress in the tumour core inhibits DC maturation, maintaining an immature phenotype in TC. The relative paucity of LA in the tumour centre in our and other studies (36,37) further drives newly recruited Immature DCs to migrate along outward CCL19/CCL21 gradients (38) and accumulate in peritumoural compartments (OM, PT) enriched in LA. LA, particularly those exhibiting features of tertiary lymphoid structures (TLS), may serve as a niche for terminal DCs maturation (37,39). CD208⁺/DC-LAMP⁺ DCs are widely recognized as a reliable immunohistochemical marker of TLS (40). In our study, we detected a high density of CD208⁺ DCs within LA. These factors collectively shape a TME that favours immature DC accumulation in TC, with maturation occurring predominantly in the tumour exterior. This interpretation is supported by our correlation analysis, which revealed the strongest associations between CD1a in TC and CD208 in OM in both pCRC and LM and confirm a unidirectional “recruitment–maturation” migration pattern. Compared between pCRC and LM, greater densities of CD1a+ DCs were observed in corresponding compartments of pCRC in synchronous disease. Synchronous LM establish a VEGF-A/IL-10/TGF-β-rich tolerogenic niche (4,41), that can suppress CCR7-mediated homing and maturation of DC precursors (42), preventing their accumulation in the metastatic nodule. Circulating monocytes are instead retained at the CD1a⁺ stage by GM-CSF, CXCL8 and CCL2 gradients in the primary tumour (43,44), turning the pCRC into a functional “monocyte reservoir”. This distribution is consistent with the concomitant immunity model (2), whereby the primary tumour serves as the main antigen source, partially restrains growth of secondary lesions, and limits influx of antigen-presenting cells into metastases. Also, greater CD208⁺ density was found in the TC of pCRC compared with LM in both cohorts. This finding suggests that the metastatic niche is less permissive for DC maturation (45) . Immunosuppressive mediators such as IL-10, TGF-β, and VEGF have been shown to inhibit DC differentiation and antigen-presenting function (41), which may explain the impaired accumulation of mature DCs in LM. We also observed differences that depended on the pattern of metastasis: in metachronous LM, CD1a⁺ density was higher in OM and CD208⁺ density was higher in IM and PT than in synchronous lesions (p<0.05), consistent with a distinct immune architecture. As reported by Wenchao Xu et al. (2025), after primary tumour resection, metastases evolve under prolonged cytokine–chemokine stimulation, where sustained low levels of GM-CSF, CXCL8 and CCL2 maintain monocyte influx, progressively expanding the CD1a⁺ pool (46). Survival analysis in synchronous and metachronous cohorts The prognostic impact of DC subsets in CRC has been widely debated, with previous studies reporting conflicting results and providing little information on synchronous versus metachronous liver metastases. Our survival analysis addresses this gap, demonstrating that the timing of metastasis is a decisive factor, with synchronous and metachronous lesions characterized by distinct DCs dynamics that critically influence patient outcomes. Thus, survival analysis demonstrated that a high density of mature CD208⁺ cells in the TC of synchronous LM was associated with nearly a two-fold reduction in the risk of death. In our cohort, LA enriched in mature CD208⁺ DCs were frequently observed in TC and IM of LM (Figure 4S). Several recent studies confirmed that TLS can develop in the centre of a metastasis. Intratumoural TLS function as local hubs for CD8⁺ T-cell recruitment, antigen presentation, clonal expansion, survival, and activation of effector T-cell, collectively generating a stronger anti-tumour immune response associated with improved free recurrence and overall survival (47). Compared with peritumoural TLS, intratumoural TLS demonstrated a stronger antitumour effect in primary and metastatic CRC and other malignancies (48) . Although current study was not aimed to characterize TLS, which would require more extensive phenotyping, the presence of mature DCs within peripheral T cell zone themselves is one of distinguishing features of TLS vs simple LA (47). Our multiple regression models revealed CD1a⁺ DCs in the TC and PT region of synchronous LM as the strongest predictors for densities of CD208+ cells in IM of LM, which, in turn, predicted their densities in the TC of LM. The high elasticity coefficient for CD1a+ DCs in the TC of LM (0.8) underscores the primary role of this cell type in establishing the pool of mature DCs within TC of LM. CD1a⁺ cells in PT likely serve as a reservoir that seeds in the TC and differentiates into mature DCs, amplifying the local immune response. This result corroborates the above concept of recruitment of immature DCs into the tumour and underscore the importance of rapid maturation of CD1a⁺ cells within intratumoural TLS in TC. We can hypothesize that presence and functional integrity of TLS within LM core, where CD1a⁺ maturate into CD208⁺, governs the clinical impact of DCs. Our results indicate that prognostic associations of DCs in CRC LM depend on DC abundance, on the distribution of immature and mature DCs across compartments, and on metastatic timing. Strength and limitations of the study DCs markers were quantitatively assessed on digitized whole slide images using specialized software, minimizing observer bias. To our knowledge, this is the first study to compare the compartment specific prognostic significance of immature CD1a⁺ and mature CD208⁺ DCs using triplicate pCRC samples. NAM and synchronous or metachronous LM, capturing the key maturation axis relevant to antigen presentation. Immune profiling of NAM alongside pCRC offers insights into TME evolution, highlights TC as a critical immune regulatory compartment, and reveals favorable prognostic associations of these DCs subsets in LM, which may inform risk stratification and immunomodulatory strategies. However, several limitations should be noted. The relatively small sample size and the lack of comprehensive molecular and mutational data for many patients limited the power to detect certain survival associations, however mutational analysis is currently ongoing in our lab. Heterogeneity of adjuvant and neoadjuvant regimens precluded a reliable assessment of interactions between therapy and the immune system. Also, to obtain a comprehensive understanding of the spatial distribution of DCs within pCRC and LM tissues, as well as to evaluate DCs migration across different compartments, future studies should employ spatial transcriptomics in combination with DCs tracking experiments. Finally, although CD1a/CD208 immunophenotyping captured the most relevant DCs maturation dynamics in this study, we did not assess other myeloid populations that may participate in interactions between the tumour and the immune system, which should be addressed in future studies. Conclusion In conclusion, this study provides the first comprehensive spatial mapping of CD1a⁺ and CD208⁺ DCs from NAM through pCRC to paired liver metastases. We identified opposing gradients of CD1a⁺ and CD208⁺ DCs consistent with an axis linking recruitment and maturation, with immature CD1a⁺ cells enriched in TC and mature CD208⁺ cells predominating in peripheral compartments. Importantly, survival analysis revealed distinct prognostic patterns depending on the timing of metastasis: in synchronous LM, higher CD208⁺ density in the TC was associated with improved OS, whereas in metachronous LM, favourable outcomes were linked to higher CD1a⁺ density in the TC. These findings indicate that patient prognosis depends not only on DC abundance but also on their compartmental distribution, with the TC playing a key role, and on the timing of metastasis. Taken together, our results highlight the clinical relevance of spatial immune profiling of DCs as a potential tool for prognostic assessment and therapeutic stratification in CRC with liver metastases. Abbreviations CRC Colorectal cancer DCs Dendritic cells FFPE Formalin fixed-paraffin embedded tissue HRs Hazard ratios IM Inner margin LA Lymphoid aggregates LF Lymphoid follicles LM Liver metastasis NAM Non tumour adjacent mucosa OM Outer margin OS Overall survival pCRC Primary colorectal cancer ROIs Regions of interests PT Peritumour zone TC Tumour center TLS Tertiary lymphoid structures TME Tumour microenvironment Declarations Conflict of interest The authors have no conflicts of interest to declare. Funding This research was funded by the grants AZV NU21 03 00506, SALVAGE project (OP JAK, reg. no. CZ.02.01.01/00/22_008/0004644) co financed by the European Union and the state budget of the Czech Republic, and by the Cooperatio Program, research area SURG. Authors' contributions Conceptualization: Kari Hemminki, Andriy Trailin; data curation: Andriy Trailin, Lenka Červenková, Filip Ambrozkiewicz, Esraa Ali, Sergii Pavlov, Wenjing Ye, Ondřej Vyčítal, and Ondrej Daum; methodology: Andriy Trailin, Lenka Červenková, Filip Ambrozkiewicz, Sergii Pavlov, Ondřej Vyčítal, and Ondrej Daum; validation: Andriy Trailin, Václav Liška and Kari Hemminki; formal analysis: Andriy Trailin, Lenka Červenková, Filip Ambrozkiewicz, Esraa Ali, Sergii Pavlov, Wenjing Ye, Ondřej Vyčítal; writing original draft: Sergii Pavlov; review and editing: Kari Hemminki, Andriy Trailin and Václav Liška; resources: Kari Hemminki and Václavn Liška; supervision: Kari Hemminki, Andriy Trailin; project administration: Kari Hemminki, Václav Liška; funding acquisition: Kari Hemminki and Václav Liška. All authors have read and agreed to the published version of the manuscript. Date availability All data generated or analyzed during this study are included in this article and its additional material files. Further enquiries can be directed to the corresponding author. Ethics approval This retrospective study was conducted in compliance with the ethical standards outlined in the Declaration of Helsinki (2013 version). The need for informed consent was waived by the Ethics 45 Committee of the Faculty of Medicine and University Hospital in Pilsen. The study was approved 46 by the Ethics Committee of the Faculty of Medicine and University Hospital in Pilsen (300/2020, 47 17 June 2020). Consent to participate Not applicable Consent for publication Not applicable. References Yu H, Hemminki K (2020) Genetic epidemiology of colorectal cancer and associated cancers. 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Supplementary Files SupplementaryTable.docx SupplementaryFigures.pdf Cite Share Download PDF Status: Published Journal Publication published 18 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 17 Oct, 2025 Reviews received at journal 17 Oct, 2025 Reviews received at journal 12 Oct, 2025 Reviewers agreed at journal 11 Oct, 2025 Reviews received at journal 01 Oct, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers invited by journal 24 Sep, 2025 Editor assigned by journal 19 Sep, 2025 Submission checks completed at journal 19 Sep, 2025 First submitted to journal 11 Sep, 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. 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1","display":"","copyAsset":false,"role":"figure","size":77046,"visible":true,"origin":"","legend":"\u003cp\u003eStatistics depicting the spatial distribution of CD1a+ and CD208+ cells per mm\u003csup\u003e2\u003c/sup\u003e in NAM and TC of pCRC in patients with synchronous metastasis (a) and metachronous metastasis (b). Black lines: medians, ****- p\u0026lt;0.0001 Abbreviations: NAM: tumour-adjacent normal mucosa, TC: tumour center, pCRC: primary colorectal cancer\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/865a1e95922151ccef615217.png"},{"id":93015747,"identity":"2dc74d80-6f0b-4a21-a959-0e1fdf1d74d6","added_by":"auto","created_at":"2025-10-08 07:59:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119991,"visible":true,"origin":"","legend":"\u003cp\u003eStatistics depicting the spatial distribution of CD1a (a, b) and CD208 (c, d) cells per mm2 in ROIs in pCRC and LM in patients with synchronous metastasis and metachronous metastasis. Black lines: medians, red lines: significant between synchronous and metachronous cohorts ROIs * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001 Abbreviations: ROI: region of interesting, LM: liver metastasis, NAM: normal mucosa, TC: tumour center, IM: inner invasive margin, OM: outer margin, PT: peritumour zone, pCRC: primary colorectal cancer.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/603e542c6cc3dbe8d25923ec.png"},{"id":93015746,"identity":"76a1ec98-bfeb-485d-aa36-a0c57fa658ae","added_by":"auto","created_at":"2025-10-08 07:59:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66096,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots of univariable hazard ratios for OS for synchronous and metachronous group.\u003c/p\u003e\n\u003cp\u003eAbbreviations: pCRC: primary colorectal cancer, LM: liver metastasis, TC: tumour center, IM: inner invasive margin, OM: outer invasive margin, PT: peritumour zone, OS- overall survival\u003c/p\u003e\n\u003cp\u003eRed line, * – statistically significant variable (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/38faad64d70369e91437ab47.png"},{"id":98814222,"identity":"46b385e7-e82e-478d-a82e-b838c1bb36b0","added_by":"auto","created_at":"2025-12-22 16:11:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1031902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/093c7cd1-450c-4543-8c74-8264a475faf0.pdf"},{"id":93015744,"identity":"f7d2bcca-cb43-4459-8192-ced7d64315fc","added_by":"auto","created_at":"2025-10-08 07:59:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":79386,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/8aebeddb24dcf40e564e6ec4.docx"},{"id":93015749,"identity":"9058a07b-edcb-4d66-8547-a5a6e37f8623","added_by":"auto","created_at":"2025-10-08 07:59:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1771290,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7588685/v1/90c0bdb99e997e6ccf9b0b8b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSpatial Patterns and Prognostic Relevance of CD1a Immature and CD208 Mature Dendritic Cells in Colorectal Cancer From Tumour-adjacent Mucosa to Liver Metastases\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is a leading cause of cancer-related deaths. Despite improved treatment, up to 60% of patients ultimately develop distant metastases, the liver being the main site because of portal drainage and a permissive pre‑metastatic niche (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The immune contexture of tumour microenvironment (TME) in both primary and metastatic tumours is now recognized as a key determinant of whether disseminated CRC cells are eliminated, held in dormancy, or allowed to progress (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the diverse immune populations shaping the CRC microenvironment, dendritic cells (DCs) represent central orchestrators of antitumour immunity (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). DCs are a highly heterogeneous cell population comprised of several subsets with distinct origins, locations, migratory and functional properties (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), which bridge innate and adaptive immune responses (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFirst subset of classical DCs (cDC1) are the principal cells for cross-presenting tumour antigens to CD8⁺ T lymphocytes and initiating cytotoxic responses, besides priming CD4⁺ T cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The cDC2 subset activates CD4⁺ T lymphocytes and drives their polarization into diverse T-helper populations (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The contribution of cDC1 and cDC2 to antitumour immunity is context-dependent and often contradictory, complicating evaluation of their prognostic impact in CRC (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Plasmacytoid DCs (pDCs) are characterized by robust production of type I interferons and, under physiological conditions, play a key role in antiviral defense (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It is known that pDCs can infiltrate solid tumours, although they are relatively scarce (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and their antitumour efficacy is limited (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Monocytic DCs (moDCs) arise from circulating monocytes during precancerous transformation and are dominant in tumour tissue (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although moDs can activate CD4⁺ and CD8⁺ T cells and cross-present antigens, dendritic-cell maturation in tumour tissue is frequently dysregulated, shifting the balance toward immature states (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). At present, CD1a is commonly accepted as a marker of monocytic (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and immature DCs (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), whereas CD208 (DC-LAMP) is recognized as a marker of mature DCs (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, the specific roles of DCs subsets and their prognostic significance in CRC progression remain only partially understood and are mostly contradictory. Several studies have reported a positive prognostic impact of CD1a\u0026thinsp;+\u0026thinsp;DCs in CRC (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), while other reports have described the immunosuppressive role of CD1a\u0026thinsp;+\u0026thinsp;DCs and their negative influence on patient survival in CRC (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Similarly, CD208\u0026thinsp;+\u0026thinsp;DCs have been associated with a favorable prognosis in CRC in (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), whereas other studies have linked it to adverse outcomes (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the gut mucosa, immature DCs constantly sample the environment by taking up antigens from the lumen through phagocytosis and macropinocytosis. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Upon maturation and migration to gut-associated lymphoid tissues, these DCs present processed antigens to T cells, which can then elicit the adaptive immune response towards pathogen clearance or tolerogenic pathways for maintaining homeostasis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, evidence remains limited on how DCs in non tumor adjacent mucosa (NAM) may influence the initiation, progression and clinical course of colorectal cancer.\u003c/p\u003e\u003cp\u003eIt remains unclear whether the spatial localization of DCs has prognostic significance in CRC. High-resolution mapping by Miller et al. revealed a sharp dichotomy between tumour center and invasive margin, with DCs density peaking in invasive margin, where high cell densities predict better survival (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Comparative studies assessing DCs distribution and prognostic impact between primary CRC and liver metastases, critical for understanding the loss of immune surveillance in metastases (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), are lacking.\u003c/p\u003e\u003cp\u003eA synthesis of literature has highlighted three major gaps: limited data on quantitative and spatial profiles of CD1a⁺ and CD208⁺ DCs in CRC from NAM through primary tumour to liver metastases; absence of studies addressing landscape of DCs in synchronous versus metachronous CRC; few, often inconsistent, cohort studies linking these subsets to patient outcomes. The aim of the present study was therefore to map the distribution of CD1a⁺ and CD208⁺ DCs from non-tumour mucosa through pCRC to LM, and to determine how compartment-specific cell counts associate with overall survival in synchronous versus metachronous cases.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003ePatients\u003c/p\u003e\u003cp\u003e All patients who underwent curative resection of pCRC followed by hepatic resection for the first recurrence of CRC at Pilsen University Hospital between 1999 and 2021 were retrospectively reviewed. LM detected at pCRC diagnosis defined the stage IV synchronous cohort (n\u0026thinsp;=\u0026thinsp;80); LM identified 17 (1\u0026ndash;59) months (median (interval) after pCRC resection defined the stage I\u0026ndash;III metachronous cohort (n\u0026thinsp;=\u0026thinsp;100).\u003c/p\u003e\u003cp\u003eInclusion criteria required first-episode LM, curative-intent surgery for both pCRC and LM, complete clinical and survival data, and available good quality formalin fixed-paraffin embedded (FFPE) tissue. Exclusion criteria were multiple primaries, pre-operative extra-hepatic disease, previous liver resections, neoadjuvant chemoradiotherapy, emergency surgery, or death within 30 days post-operation. Ninety-nine patients fulfilled all criteria (55 with synchronous LM and 44 with metachronous LM).\u003c/p\u003e\u003cp\u003eDemographic, pathological and clinical variables were extracted (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Tumours were staged according to the criteria of AJCC 8th edition; most were histological type NOS, grade 2. The cohorts differed only in median LM size (larger in the metachronous group) and in the proportion of patients receiving adjuvant FOLFOX (lower in the metachronous group). The study followed the Declaration of Helsinki (2013) and was approved by the local ethics committee (300/2020, 17 June 2020).\u003c/p\u003e\u003cp\u003ePathology and immunohistology\u003c/p\u003e\u003cp\u003eFFPE tissues of pCRC, LM and NAM from each patient were identified and cut into 4-\u0026micro;m sections. Sample for NAM was taken from the resection margin (oral or aboral), which was the closest to the tumour, with a median distance from the tumour 34 mm (range: 4-200 mm). In case of multiple LMs, we selected the metastatic tumour with the least regressive changes. One or two tissue sections were mounted onto BOND Plus Microscope Slides (Cat#00270, Leica Biosystems Newcastle Ltd., Newcastle, UK). Immunohistochemical detection of CD1a and CD208 cells was performed using fully automated BOND RXm IHC/ISH stainer. Ready-to-use monoclonal primary antibodies for CD1a (clone MTB1) from Leica Biosystems (Newcastle Ltd., United Kingdom), CD208 (clone EPR24265-8) from Abcam (Abcam Ltd., United Kingdom) were used. Binding of primary antibodies with their targets was visualized using horseradish peroxidase (HRP)-linker antibody conjugate system (Bond\u0026trade; Polymer Refine Detection). Sections were counterstained with Mayer's haematoxylin and embedded into Micromount mounting medium (Leica Biosystems Newcastle Ltd., United Kingdom). Appropriate positive (tonsils) and negative tissue control samples were used throughout.\u003c/p\u003e\u003cp\u003eImage analysis\u003c/p\u003e\u003cp\u003eWhole-slide scans were acquired using an Olympus VS200 scanner (Olympus, Shinjuku, Japan). Regions of interest (ROIs) in pCRC and LM, including tumour center (TC), inner margin (IM), outer margin (OM), and peritumour zone (PT), were annotated and analysed in QuPath v.0.4.3 using custom scripts (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/sergii01-cuni/script_zones\u003c/span\u003e\u003cspan address=\"https://github.com/sergii01-cuni/script_zones\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). NAM was annotated as a single region above the muscularis mucosa, encompassing surface epithelium, intestinal crypts, and lamina propria, with exclusion of dysplasia, crypt lumina, and artefacts. Tumour borders were defined at the interface between malignant cell nests and adjacent non-tumour tissue, excluding luminal surface, large vessels, normal mucosa, dysplastic epithelium, muscularis propria, supportive stroma\u0026thinsp;\u0026gt;\u0026thinsp;2 mm, extracellular mucin, fat, necrosis, abscesses, haemorrhages, and artefacts. CD1a⁺ and CD208⁺ cell densities were calculated for NAM and each ROI as the number of immunopositive cell profiles per total ROI area. To eliminate skewness in the distribution, the raw data were converted into percentile values for survival analysis and then categorized into two groups: low (below the 25th percentile) and high (25th\u0026ndash;100th percentile).\u003c/p\u003e\u003cp\u003eFollow-up\u003c/p\u003e\u003cp\u003eFollow-up continued through December 2023. Median surveillance after liver metastasectomy was 84 months (95% CI 5\u0026ndash;163) in the synchronous cohort and 61 months (95% CI 54\u0026ndash;68) in the metachronous cohort. Patients were reviewed every three months for two years, then semi-annually; each visit included tumour-marker assays, chest X-ray, abdominal ultrasound, and CT. PET or MRI was added at the multidisciplinary team\u0026rsquo;s discretion.\u003c/p\u003e\u003cp\u003eOutcomes\u003c/p\u003e\u003cp\u003eThe endpoint of the study was overall survival (OS), measured from resection of LM to death from any cause. Patients without death were censored at their last follow-up. OS after LM surgery were not statistically different between groups (data not shown).\u003c/p\u003e\u003cp\u003eStatistical methods\u003c/p\u003e\u003cp\u003eContinuous variables with non-normal distributions were presented as median (range) and compared using the Mann\u0026ndash;Whitney U test or, for repeated measures, Friedman ANOVA with Wilcoxon matched-pairs tests (Bonferroni adjusted). Categorical variables were expressed as counts (%). OS was estimated by Kaplan\u0026ndash;Meier analysis and compared with the log-rank test; prognostic value of predictors was assessed by Cox regression with hazard ratios (HRs) for high vs. low groups. Multiple regression with backward stepwise elimination identified predictors of DCs density in ROIs; model quality was assessed by R\u0026sup2; and Fisher\u0026rsquo;s F test, with elasticity coefficients quantifying covariate influence. Associations between variables were tested by Spearman correlation. Analyses used GraphPad Prism 9.0; survival modelling employed the finalfit package, with significant Cox results visualised in survival and survminer. Two-sided p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e1. Morphology and topography of CD1a⁺ and CD208⁺ DCs in NAM, pCRC and LM\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eRounded CD1a⁺ DCs were extremely rare in the lamina propria of NAM; in addition, they were occasionally present within the marginal zone of lymphoid follicles (LF) associated with NAM (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Rounded and stellate-shaped CD208⁺ DCs were larger, they spanned the lamina propria forming micro-clusters of 10\u0026ndash;20 cells (Fig. 1Sb) and also accumulated in mantle and marginal zones of LF (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eIn pCRC, CD1a⁺ cells were sparse, confined to stromal spaces between glands (Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003ea) with slightly higher density at the invasive front and sporadically dispersed in PT and OM. CD208⁺ cells were abundant in the stromal regions surrounding the tumour and within lymphoid aggregates (LA) (Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eb), which were more frequent toward the PT region.\u003c/p\u003e\n\u003cp\u003eIn LM, CD1a⁺ DCs were exceptionally scarce, appearing as solitary elements in stroma and LA (Fig.\u0026nbsp;3Sa). CD208⁺ DCs were abundant, forming micro-clusters in the TC and IM, accumulating predominantly in the LA, which were, however, more numerous in the OM (Fig.\u0026nbsp;3Sb).\u003c/p\u003e\n\u003cp\u003eTC and IM of LM were also enriched in LA (Fig.\u0026nbsp;4Sa-d), which were more frequent than in pCRC, lying mainly at the tumour\u0026ndash;liver interface. Collectively, LM thus display a pronounced imbalance between scant and dispersed immature CD1a⁺ cells and spatially organized clusters of mature CD208⁺ DCs.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e2. Distribution of CD1a\u0026thinsp;+\u0026thinsp;and CD208\u0026thinsp;+\u0026thinsp;DCs between TC of pCRC and NAM\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of CD1a⁺ cell distribution in NAM and in the TC of pCRC showed significantly greater cell densities in the TC of pCRC in both cohorts (Fig. 1a, b). By contrast, the density of CD208⁺ DCs was not statistically significant different between the two tissues in either group (Fig. 1a, b).\u003c/p\u003e\n\u003cp\u003eThe number of CD208⁺ cells within NAM was significantly higher than the number of CD1a cells in both patient groups (p\u0026thinsp;=\u0026thinsp;0.0001). No statistically significant difference in densities of either CD1a⁺ or CD208⁺ cells in NAM were observed between groups.\u003c/p\u003e\n\u003cp\u003eWe found significant correlation between densities of CD1a\u0026thinsp;+\u0026thinsp;and CD208\u0026thinsp;+\u0026thinsp;DC in the TC, and between CD1a\u0026thinsp;+\u0026thinsp;DC in the TC and NAM in the cohort with synchronous metastasis (Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e), whereas in patients with metachronous metastasis densities of CD1a\u0026thinsp;+\u0026thinsp;DC and CD208\u0026thinsp;+\u0026thinsp;DC correlated only in the TC (Table S3).\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e3. Distribution of CD1a and CD208 cells in pCRC and LM\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eIn pCRC, CD1a⁺ density was highest at the IM, following the order IM\u0026thinsp;\u0026gt;\u0026thinsp;TC\u0026thinsp;\u0026gt;\u0026thinsp;OM\u0026thinsp;\u0026gt;\u0026thinsp;PT in synchronous cases and IM\u0026thinsp;\u0026asymp;\u0026thinsp;TC\u0026thinsp;\u0026gt;\u0026thinsp;OM\u0026thinsp;\u0026gt;\u0026thinsp;PT in metachronous cases (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). In LM, both cohorts showed order IM\u0026thinsp;\u0026gt;\u0026thinsp;TC\u0026thinsp;\u0026gt;\u0026thinsp;OM\u0026thinsp;\u0026gt;\u0026thinsp;PT (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), with greater density in OM of synchronous LM than metachronous LM (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eCD208⁺ cells in pCRC displayed OM\u0026thinsp;\u0026gt;\u0026thinsp;PT\u0026thinsp;=\u0026thinsp;TC\u0026thinsp;\u0026ge;\u0026thinsp;IM order in both cohorts, with no intergroup differences; in LM the pattern was OM\u0026thinsp;\u0026gt;\u0026thinsp;PT\u0026thinsp;\u0026gt;\u0026thinsp;TC\u0026thinsp;=\u0026thinsp;IM, but IM and PT of metachronous LM contained more CD208⁺ cells than synchronous LM (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). CD208⁺ cells predominated over CD1a⁺ in OM and PT of both pCRC and LM in both cohorts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas in synchronous disease CD1a⁺ cells exceeded CD208⁺ in pCRC IM (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and LM TC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eCompared between pCRC and LM, CD1a⁺ density was higher in every pCRC compartment than in the corresponding compartment of LM in the synchronous cohort (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while CD208⁺ density was greater in TC of pCRC in both cohorts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eSpearmanʼs analysis revealed significant correlations for CD1a⁺ and CD208⁺ DCs between all ROIs within pCRC and within LM (Table S3, S4), with the strongest links consistently observed between adjacent ROIs (TC and IM, IM and OM, OM and PT). In synchronous pCRC, the strongest correlations involved CD1a TC with CD208 IM and TC, whereas in LM CD1a TC correlated with CD208 OM. In metachronous pCRC, CD1a IM correlated with CD208 PT, IM, and OM, whereas in LM the key associations were CD1a TC with CD208 OM and TC. No significant correlations were found between corresponding ROIs of pCRC and LM, except for CD1a in OM (p\u0026thinsp;=\u0026thinsp;0.05) in metachronous cases.\u003c/p\u003e\n\u003ch3\u003eSurvival analysis\u003c/h3\u003e\n\u003cp\u003eSurvival analysis demonstrated that higher CD208⁺ cell density in the TC of synchronous LM was significantly associated with reduced mortality risk (HR\u0026thinsp;=\u0026thinsp;0.47; 95% CI: 0.23\u0026ndash;0.94; p\u0026thinsp;=\u0026thinsp;0.033). In contrast, increased CD1a⁺ density in the TC of metachronous LM was associated with improved survival (HR\u0026thinsp;=\u0026thinsp;0.44; 95% CI: 0.19\u0026ndash;1.00; p\u0026thinsp;=\u0026thinsp;0.050), although the association was borderline.\u003c/p\u003e\u003cp\u003eKaplan\u0026ndash;Meier analysis confirmed association of CD208⁺ cells in the TC of synchronous LM with longer OS (Figure S5a) with the curve showing consistently higher survival across the entire observation period. CD1a⁺ cell density in the TC of metachronous LM were significantly associated with a longer OS (Figure S5b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on survival analysis results, multiple linear regression was used to identify independent predictors of CD208⁺ DCs density in TC of synchronous LM and CD1a⁺ DC density in TC of metachronous LM. No valid model was obtained for CD1a, whereas in synchronous LM a robust model identified CD208 in the IM of LM as the strongest predictor (Model 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD208 TC of LM =1,753+0,475\u0026times;CD208 IM of LM\u0026nbsp;\u0026nbsp;\u003c/strong\u003e(\u003cem\u003eModel 1\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e=0.533, p\u0026lt;0.0001\u003c/p\u003e\n\u003cp\u003eThe model was statistically significant; an increase of 10 cells in CD208 in the IM of LM corresponds to a rise of roughly 5 CD208+ cells in the TC of LM. Model stability was supported by a high R\u0026sup2; and a significant F-test p-value.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccordingly, we constructed a model predicting density of CD208+ DC in IM of synchronous LM:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD208 IM of LM = 1.696 \u0026times; CD1a TC of LM \u0026minus; 0.180 \u0026times; CD1a IM of LM + 2.680 \u0026times; CD1a PT of LM\u0026nbsp;\u003c/strong\u003e(\u003cem\u003eModel 2\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eR\u0026sup2; = 0.679, p \u0026lt; 0.001\u003c/p\u003e\n\u003cp\u003eThe model 2 was statistically significant and demonstrated high quality. CD1a in the TC and PT region of LM had a positive effect on density of CD208 in the IM of LM whereas CD1a in the IM of LM had a negative effect.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo quantify the relative impact of each variable, elasticity coefficients were calculated (Table S5). As shown in Table 1, density of CD1a+ DCs in the TC of LM had the strongest effect on density of CD208+ DCs in the IM of LM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociations of clinical and pathology variables with CD1a and CD208 DCs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTables S6\u0026ndash;S8 summarize associations between clinicopathological variables and CD1a, CD208 DC densities across NAM, pCRC and LM, DCs and chemotherapy administered before and after liver resection, and DCs and FOLFOX-based chemotherapy, respectively. Statistically significant differences are indicated in the Table S6-8 legends.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe immune landscape of pCRC and its metastases constitutes a dynamic system in which DCs are one the main actors of the immune response. Most previous studies have investigated DCs primarily in pCRC, often as a pooled population or restricted to the tumour core, without considering their maturation state or compartment-specific localization; in contrast, studies focusing on liver metastases are relatively few and provide only limited insight into DC heterogeneity (15,30\u0026ndash;32), To our knowledge, this is the first study to comprehensively characterize CD1a⁺ and CD208⁺ DCs distribution across NAM and several anatomical compartments in both primary CRC and paired synchronous or metachronous LM, and to directly relate these spatial patterns to patient survival.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of CD1a\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CD208 DCs in NAM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have provided very limited information on the landscape of immature and mature DCs in NAM, as most investigations have concentrated on tumour tissue itself. However, establishing a baseline profile of DCs subsets in NAM could serve as a reference for assessing tumour-associated alterations. We observed high densities of CD208⁺ DCs with an almost complete absence of CD1a⁺ DCs within NAM, yielding a CD208/CD1a ratio of approximately 25:1. Mature CD208⁺ DCs in NAM were predominantly located within mucosa-associated LF, where DCs complete functional maturation and participate in antigen presentation \u003cspan lang=\"EN-US\"\u003e(33)\u003c/span\u003e. Absence of correlation between CD208⁺ DCs in NAM and CD1a+ or CD208+ cells across different ROIs of pCRC suggest their autonomous resident profile in NAM, which is supported by the literature\u0026nbsp;(27).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of CD1a\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CD208 DCs in pCRC and LM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost earlier studies have investigated DCs either as a pooled population or with an emphasis on the tumour core of primary or metastatic tumour, while comparative analyses between primary tumours and paired liver metastases, including fine regional assessment, remain scarce. Greater densities of CD1a+ DCs in TC of pCRC vs NAM can reflect CCL2-dependent influx of circulating monocytes that differentiate into moDCs within the TME (27,34), supplying therefore precursors for peritumoural maturation. Similar mechanism can be responsible for higher densities of CD1a+ DCs in the tumour interior of LM vs tumour exterior. Across pCRC and LM tissue in both synchronous and metachronous cohort of patients, CD1a⁺ density decreased from the TC/IM towards the OM/PT, whereas CD208⁺ density followed the opposite gradient. This consistent trend aligns with mechanisms described by Jie Chen, Yuhang Duan et al. (2023) (35), whereby hypoxic stress in the tumour core inhibits DC maturation, maintaining an immature phenotype in TC. The relative paucity of LA in the tumour centre in our and other studies (36,37) further drives newly recruited Immature DCs to migrate along outward CCL19/CCL21 gradients (38) and accumulate in peritumoural compartments (OM, PT) enriched in LA. LA, particularly those exhibiting features of tertiary lymphoid structures (TLS), may serve as a niche for terminal DCs maturation\u0026nbsp;(37,39). CD208⁺/DC-LAMP⁺ DCs are widely recognized as a reliable immunohistochemical marker of TLS\u0026nbsp;(40). In our study, we detected a high density of CD208⁺ DCs within LA.\u003c/p\u003e\n\u003cp\u003eThese factors collectively shape a TME that favours immature DC accumulation in TC, with maturation occurring predominantly in the tumour exterior. This interpretation is supported by our correlation analysis, which revealed the strongest associations between CD1a in TC and CD208 in OM in both pCRC and LM and confirm a unidirectional \u0026ldquo;recruitment\u0026ndash;maturation\u0026rdquo; migration pattern.\u003c/p\u003e\n\u003cp\u003eCompared between pCRC and LM, greater densities of CD1a+ DCs were observed in corresponding compartments of pCRC in synchronous disease. Synchronous LM establish a VEGF-A/IL-10/TGF-\u0026beta;-rich tolerogenic niche\u0026nbsp;(4,41), that can suppress CCR7-mediated homing and maturation of DC precursors (42), preventing their accumulation in the metastatic nodule. Circulating monocytes are instead retained at the CD1a⁺ stage by GM-CSF, CXCL8 and CCL2 gradients in the primary tumour (43,44), turning the pCRC into a functional \u0026ldquo;monocyte reservoir\u0026rdquo;. This distribution is consistent with the concomitant immunity model (2), whereby the primary tumour serves as the main antigen source, partially restrains growth of secondary lesions, and limits influx of antigen-presenting cells into metastases. Also, greater CD208⁺ density was found in the TC of pCRC compared with LM in both cohorts. This finding suggests that the metastatic niche is less permissive for DC maturation \u003cspan lang=\"EN-US\"\u003e(45)\u003c/span\u003e. Immunosuppressive mediators such as IL-10, TGF-\u0026beta;, and VEGF have been shown to inhibit DC differentiation and antigen-presenting function (41), which may explain the impaired accumulation of mature DCs in LM.\u003c/p\u003e\n\u003cp\u003eWe also observed differences that depended on the pattern of metastasis: in metachronous LM, CD1a⁺ density was higher in OM and CD208⁺ density was higher in IM and PT than in synchronous lesions (p\u0026lt;0.05), consistent with a distinct immune architecture. As reported by Wenchao Xu et al. (2025), after primary tumour resection, metastases evolve under prolonged cytokine\u0026ndash;chemokine stimulation, where sustained low levels of GM-CSF, CXCL8 and CCL2 maintain monocyte influx, progressively expanding the CD1a⁺ pool (46).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival analysis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;synchronous\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;metachronous cohorts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe prognostic impact of DC subsets in CRC has been widely debated, with previous studies reporting conflicting results and providing little information on synchronous versus metachronous liver metastases. Our survival analysis addresses this gap, demonstrating that the timing of metastasis is a decisive factor, with synchronous and metachronous lesions characterized by distinct DCs dynamics that critically influence patient outcomes. Thus, survival analysis demonstrated that a high density of mature CD208⁺ cells in the TC of synchronous LM was associated with nearly a two-fold reduction in the risk of death. In our cohort, LA enriched in mature CD208⁺ DCs were frequently observed in TC and IM of LM (Figure 4S). Several recent studies confirmed that TLS can develop in the centre of a metastasis. Intratumoural TLS function as local hubs for CD8⁺ T-cell recruitment, antigen presentation, clonal expansion, survival, and activation of effector T-cell, collectively generating a stronger anti-tumour immune response associated with improved free recurrence and overall survival (47). Compared with peritumoural TLS, intratumoural TLS demonstrated a stronger antitumour effect in primary and metastatic CRC and other malignancies \u003cspan lang=\"EN-US\"\u003e(48)\u003c/span\u003e. Although current study was not aimed to characterize TLS, which would require more extensive phenotyping, the presence of mature DCs within peripheral T cell zone themselves is one of distinguishing features of TLS vs simple LA (47).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur multiple regression models revealed CD1a⁺ DCs in the TC and PT region of synchronous LM as the strongest predictors for densities of CD208+ cells in IM of LM, which, in turn, predicted their densities in the TC of LM. The high elasticity coefficient for CD1a+ DCs in the TC of LM (0.8) underscores the primary role of this cell type in establishing the pool of mature DCs within TC of LM. CD1a⁺ cells in PT likely serve as a reservoir that seeds in the TC and differentiates into mature DCs, amplifying the local immune response. This result corroborates the above concept of recruitment of immature DCs into the tumour and underscore the importance of rapid maturation of CD1a⁺ cells within intratumoural TLS in TC. \u0026nbsp;We can hypothesize that presence and functional integrity of TLS within LM core, where CD1a⁺ maturate into CD208⁺, governs the clinical impact of DCs.\u003c/p\u003e\n\u003cp\u003eOur results indicate that prognostic associations of DCs in CRC LM depend on DC abundance, on the distribution of immature and mature DCs across compartments, and on metastatic timing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrength and limitations of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDCs markers were quantitatively assessed on digitized whole slide images using specialized software, minimizing observer bias. To our knowledge, this is the first study to compare the compartment specific prognostic significance of immature CD1a⁺ and mature CD208⁺ DCs using triplicate pCRC samples. NAM and synchronous or metachronous LM, capturing the key maturation axis relevant to antigen presentation. Immune profiling of NAM alongside pCRC offers insights into TME evolution, highlights TC as a critical immune regulatory compartment, and reveals favorable prognostic associations of these DCs subsets in LM, which may inform risk stratification and immunomodulatory strategies.\u003c/p\u003e\n\u003cp\u003eHowever, several limitations should be noted. The relatively small sample size and the lack of comprehensive molecular and mutational data for many patients limited the power to detect certain survival associations, however mutational analysis is currently ongoing in our lab. Heterogeneity of adjuvant and neoadjuvant regimens precluded a reliable assessment of interactions between therapy and the immune system. Also, to obtain a comprehensive understanding of the spatial distribution of DCs within pCRC and LM tissues, as well as to evaluate DCs migration across different compartments, future studies should employ spatial transcriptomics in combination with DCs tracking experiments. Finally, although CD1a/CD208 immunophenotyping captured the most relevant DCs maturation dynamics in this study, we did not assess other myeloid populations that may participate in interactions between the tumour and the immune system, which should be addressed in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study provides the first comprehensive spatial mapping of CD1a⁺ and CD208⁺ DCs from NAM through pCRC to paired liver metastases. We identified opposing gradients of CD1a⁺ and CD208⁺ DCs consistent with an axis linking recruitment and maturation, with immature CD1a⁺ cells enriched in TC and mature CD208⁺ cells predominating in peripheral compartments. Importantly, survival analysis revealed distinct prognostic patterns depending on the timing of metastasis: in synchronous LM, higher CD208⁺ density in the TC was associated with improved OS, whereas in metachronous LM, favourable outcomes were linked to higher CD1a⁺ density in the TC. These findings indicate that patient prognosis depends not only on DC abundance but also on their compartmental distribution, with the TC playing a key role, and on the timing of metastasis. Taken together, our results highlight the clinical relevance of spatial immune profiling of DCs as a potential tool for prognostic assessment and therapeutic stratification in CRC with liver metastases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCRC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eColorectal cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDCs\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDendritic cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFFPE\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFormalin fixed-paraffin embedded tissue\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHRs\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHazard ratios\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eIM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInner margin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLymphoid aggregates\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLymphoid follicles\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLiver metastasis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNAM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNon tumour adjacent mucosa\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eOM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOuter margin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eOS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOverall survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003epCRC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePrimary colorectal cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eROIs\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRegions of interests\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003ePT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePeritumour zone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumour center\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTLS\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTertiary lymphoid structures\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTME\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumour microenvironment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the grants AZV NU21 03 00506, SALVAGE project (OP JAK, reg. no. CZ.02.01.01/00/22_008/0004644) co financed by the European Union and the state budget of the Czech Republic, and by the Cooperatio Program, research area SURG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Kari Hemminki, Andriy Trailin; data curation: Andriy Trailin, Lenka Červenkov\u0026aacute;, Filip Ambrozkiewicz, Esraa Ali, Sergii Pavlov, Wenjing Ye, Ondřej Vyč\u0026iacute;tal, and Ondrej Daum; methodology: Andriy Trailin, Lenka Červenkov\u0026aacute;, Filip Ambrozkiewicz, Sergii Pavlov, Ondřej Vyč\u0026iacute;tal, and Ondrej Daum; validation: Andriy Trailin, V\u0026aacute;clav Li\u0026scaron;ka and Kari Hemminki; formal analysis: Andriy Trailin, Lenka Červenkov\u0026aacute;, Filip Ambrozkiewicz, Esraa Ali, Sergii Pavlov, Wenjing Ye, Ondřej Vyč\u0026iacute;tal; writing original draft: Sergii Pavlov; review and editing: Kari Hemminki, Andriy Trailin and V\u0026aacute;clav Li\u0026scaron;ka; resources: Kari Hemminki and V\u0026aacute;clavn Li\u0026scaron;ka; supervision: Kari Hemminki, Andriy Trailin; project administration: Kari Hemminki, V\u0026aacute;clav Li\u0026scaron;ka; funding acquisition: Kari Hemminki and V\u0026aacute;clav Li\u0026scaron;ka. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDate availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its additional material files. Further enquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study was conducted in compliance with the ethical standards outlined in the Declaration of Helsinki (2013 version). The need for informed consent was waived by the Ethics 45 Committee of the Faculty of Medicine and University Hospital in Pilsen. The study was approved 46 by the Ethics Committee of the Faculty of Medicine and University Hospital in Pilsen (300/2020, 47 17 June 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu H, Hemminki K (2020) Genetic epidemiology of colorectal cancer and associated cancers. Mutagenesis 35(3):207\u0026ndash;219\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrailin A, Ali E, Ye W, Pavlov S, Červenkov\u0026aacute; L, Vyč\u0026iacute;tal O et al (2025) Prognostic assessment of T-cells in primary colorectal cancer and paired synchronous or metachronous liver metastasis. 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Front Oncol. ;15\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"colorectal cancer, synchronous and metachronous liver metastases, dendritic cells, CD1a, CD208, spatial immune profiling, overall survival","lastPublishedDoi":"10.21203/rs.3.rs-7588685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7588685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e. The prognostic role of dendritic cells (DCs) in colorectal cancer (CRC) and paired liver metastases (LM) remains unclear, particularly regarding the dynamics of immature CD1a⁺ and mature CD208⁺ subsets across anatomical compartments and synchronous versus metachronous disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and methods\u003c/strong\u003e. This retrospective cohort included patients undergoing resection of primary CRC (pCRC) and synchronous LM (N = 55) or metachronous LM (N = 44). Immunohistochemical staining for CD1a and CD208 was performed on non tumour adjacent normal mucosa (NAM), tumour center (TC), inner and outer invasive margins (IM, OM), and peritumoural tissue (PT). Cell densities were quantified on whole-slide images using QuPath software and correlated with overall survival (OS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e. CD1a⁺ DCs were nearly absent in NAM but enriched in pCRC TC, whereas CD208⁺ DCs predominated in NAM lymphoid aggregates and peripheral compartments of pCRC and LM. CD1a⁺ cells followed a TC/IM \u0026gt; OM/PT gradient, while CD208⁺ cells showed the opposite, consistent with a recruitment–maturation axis. In synchronous cases, CD1a⁺ densities were higher in pCRC than LM, supporting the role of the primary tumour as a “monocyte reservoir.” Survival analysis revealed that high CD208⁺ density in TC of synchronous LM (HR = 0.47; p = 0.033) and high CD1a⁺ density in TC of metachronous LM (HR = 0.44; p = 0.050) were both associated with reduced mortality risk.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e. This study provides the first detailed mapping of CD1a⁺ and CD208⁺ DCs across NAM, pCRC and paired LM, indicating that their prognostic impact is determined not only by absolute numbers but, more importantly, by compartmental distribution and the timing of metastasis.\u003c/p\u003e","manuscriptTitle":"Spatial Patterns and Prognostic Relevance of CD1a Immature and CD208 Mature Dendritic Cells in Colorectal Cancer From Tumour-adjacent Mucosa to Liver Metastases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 07:59:53","doi":"10.21203/rs.3.rs-7588685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-17T08:42:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-17T07:18:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T01:59:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119411241590008729022432342541431821692","date":"2025-10-12T01:27:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T10:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210684024566196816643550697497163512667","date":"2025-09-26T07:53:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196263169108996199948023181589204764687","date":"2025-09-25T06:20:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T03:12:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T09:27:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T09:26:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-09-11T06:54:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a99dc617-7679-44f2-a3d4-866a060bf35d","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:07:01+00:00","versionOfRecord":{"articleIdentity":"rs-7588685","link":"https://doi.org/10.1007/s00262-025-04238-2","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-12-18 15:58:40","publishedOnDateReadable":"December 18th, 2025"},"versionCreatedAt":"2025-10-08 07:59:53","video":"","vorDoi":"10.1007/s00262-025-04238-2","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04238-2","workflowStages":[]},"version":"v1","identity":"rs-7588685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7588685","identity":"rs-7588685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}