Bo-Wen Zheng

Background: Tumor budding (TB) is a well-established prognostic indicator in various epithelial malignancies. Chordoma, although a rare mesenchymal tumor, paradoxically exhibits prominent epithelial-like characteristics, as demonstrated in previous studies. In particular, it remains unclear whether TB-like (TBL) structures are present in chordoma, as well as the molecular mechanisms driving their formation and their functional impact on tumor progression, representing a critical gap in current knowledge. Methods: Tumor budding-like grades were defined and evaluated in tumor specimens from 481 chordoma patients across 4 large cohorts using hematoxylin-eosin and immunohistochemical staining. Multi-omics profiling, encompassing GeoMx digital spatial profiling, spatial transcriptomics, bulk RNA sequencing, single-cell RNA sequencing, single-cell ATAC sequencing, and multiplex quantitative immunofluorescence, was integrated to delineate TBL cell subpopulations (TBLCs) and their interactions with cholesterol-metabolic tumor-associated macrophages (CM-TAMs). Organoid models and in vitro/in vivo functional assays were employed for mechanistic investigation and validation. Results: Tumor budding-like structures were prevalent in chordoma, and higher TBL grades were associated with unfavorable clinical outcomes and aggressive phenotypes. Mechanistically, BACH1 in CM-TAMs drove ANGPTL4 secretion, which targeted the SDC4 receptor on TBLCs, thereby enhancing stem-like properties, promoting cholesterol accumulation, and accelerating malignant progression. Pharmacological inhibition of cholesterol metabolism or disruption of the BACH1-ANGPTL4-SDC4 signaling axis markedly reduced tumor invasiveness in both preclinical models and chordoma organoids. Conclusions: BACH1-driven CM-TAMs activate TBLCs via the ANGPTL4-SDC4 signaling axis, promoting stemness and cholesterol accumulation, ultimately driving malignant progression in chordoma. These findings uncover a previously unrecognized tumor-immune-metabolic interaction and suggest potential therapeutic targets for this disease.

Given the lack of reliable indicators for predicting prognosis and treatment response in giant cell tumor of bone (GCTB) patients, this study aimed to identify new prognostic factors by analyzing the effect of tumor‐associated macrophages (TAMs) on prognosis and denosumab treatment responsiveness. The expression of CD68⁺TAMs, CD163⁺TAMs, and IRF8⁺TAMs was detected using polychromatic fluorescence immunohistochemistry in 162 GCTB samples. TAM density was quantified through computer‐aided image analysis, and spatial parameters, including nearest neighbor distance (NND) and effective percentage (EP), were measured using HALO software. Results showed that higher densities of CD68⁺ and CD163⁺ TAMs were significantly associated with inferior progression‐free survival (PFS). A smaller NND was linked to shorter PFS. Additionally, higher CD68⁺ EP was associated with poorer PFS, whereas higher CD163⁺ EP correlated with better PFS. Receiver operating characteristic curve analysis demonstrated that TAM parameters' predictive performance was comparable to Campanacci and surgical approach in three subgroups. The ineffective denosumab‐treated group had significantly higher TAMs EP compared to the effective group. In conclusion, TAMs significantly influence the prognosis of GCTB patients and are correlated with certain invasive tumor phenotypes. Elevated TAMs levels may be associated with reduced efficacy of denosumab treatment.

Cancer‐associated fibroblasts (CAFs) constitute the primary cellular component of the stroma in chordomas, characterized by an abundance of mucinous stromal elements, potentially facilitating their initiation and progression; however, this inference has yet to be fully confirmed. In this study, single‐cell RNA sequencing (scRNA‐seq), spatial transcriptomics (ST), bulk RNA‐seq, multiplexed quantitative immunofluorescence (QIF), and in vivo and in vitro experiments were performed to determine the heterogeneity, spatial distribution, and clinical significance of CAFs in chordoma. ScRNA‐seq was performed on 87,693 single cells derived from seven tumor samples and four control nucleus pulposus samples. A distinct CAF cluster distinguished by the upregulated expression of inflammatory genes and enriched functionality in activating inflammation‐associated cells was identified. Pseudotime trajectory and cell communication analyses suggested that this inflammatory CAF (iCAF) subset originated from normal fibroblasts and interacted extensively with tumors and various other cell types. By integrating the scRNA‐seq results with ST, the presence of iCAF in chordoma tissue was further confirmed, indicating their positioning at a distance from the tumor cells. Bulk RNA‐seq data analysis from 126 patients revealed a correlation between iCAF signature scores, chordoma invasiveness, and poor prognosis. QIF validation involving an additional 116 patients found that although iCAFs were not in close proximity to tumor cells compared with other CAF subsets, their density correlated with malignant tumor phenotypes and adverse outcomes. In vivo and in vitro experiments further confirmed that iCAFs accelerate the malignant progression of chordomas. These findings could provide insights into the development of novel therapeutic strategies. © 2024 The Pathological Society of Great Britain and Ireland.

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Background: Currently, tumor budding (TB) is defined as an important factor for a poor prognosis in various types of cancers. The authors identified a significant presence of TB‐like structures at the tumor invasive front in giant cell tumor of bone (GCTB), which may have the same biologic function as TB. The objective of this report was to describe the distribution of TB in GCTB and investigate its correlation with clinicopathologic characteristics, the immune microenvironment, survival prognosis, and response to denosumab treatment.Methods: This multicenter cohort study included 426 patients with GCTB who received treatment between 2012 and 2021 at four centers. Two independent pathologists performed visual assessments of TBL structures in hematoxylin‐and‐eosin–stained tumor sections. Immunohistochemistry was used to evaluate tumor‐infiltrating lymphocyte subtypes (CD3‐positive, CD4‐positive, CD8‐positive, CD20‐positive, programmed cell death protein‐1–positive, programmed cell death‐ligand 1positive, and FoxP3‐positive) as well as Ki‐67 expression levels in 426 tissue samples. These parameters were then analyzed for associations with patient outcomes (local recurrence‐free survival [LRFS] and overall survival [OS]), clinicopathologic characteristics, and response to denosumab treatment.Results: High‐grade TB was associated with poorer LRFS and OS in both patient groups. In addition, TB was correlated with various clinicopathologic features, tumor‐infiltrating lymphocyte expression, and response to denosumab treatment. TB outperformed the traditional Enneking and Campanacci staging systems in predicting patient LRFS and OS.Conclusions: The current data support the assessment of TBL structures as a reliable prognostic tool in GCTB, potentially aiding in the development of personalized treatment strategies for patients.

Currently, the oncogenic mechanism of endoplasmic reticulum stress‐CAF (ERS‐CAF) subpopulation in chordoma remains unknown. Here, single‐cell RNA sequencing, spatial transcriptomics, GeoMx Digital Spatial Profiler, data‐independent acquisition proteomics, bulk RNA‐seq, and multiplexed quantitative immunofluorescence are used to unveil the precise molecular mechanism of how ERS‐CAF affected chordoma progression. Results show that hypoxic microenvironment reprograms CAFs into ERS‐CAF subtype. Mechanistically, this occurrs via hypoxia‐mediated transcriptional upregulation of IER2. Overexpression of IER2 in CAFs promotes chordoma progression, which can be impeded by IER2 knockdown or use of ERS inhibitors. IER2 also induces expression of ERS‐CAF marker genes and results in production of a pro‐tumorigenic paracrine GMFG signaling, which exert its biological function via directly binding to ITGB1 on tumor cells. ITGB1 inhibition attenuates tumor malignant progression, which can be partially reversed by exogenous GMFG intervention. Further analyses reveal a positive correlation between ITGB1^high tumor cell counts and SPP1⁺ macrophage density, as well as the spatial proximity of these two cell types. Clinically, a significant correlation of high IER2/ITGB1 expression with tumor aggressive phenotype and poor patient survival is observed. Collectively, the findings suggest that ERS‐CAF regulates SPP1⁺ macrophage to aggravate chordoma progression via the IER2/GMFG/ITGB1 axis, which may be targeted therapeutically in future.

PURPOSE: The prognostic value of lymphocyte infiltration score (LIS) and its nearest neighbor distance to tumor cells (NNDTC) in giant cell tumor of bone (GCTB) is currently not well established. This study aims to characterize LIS and NNDTC and examine their correlation with denosumab treatment responsiveness, clinicopathologic features, and patient prognosis. METHODS: Using multiplexed quantitative immunofluorescence, LIS was evaluated in 253 tumor specimens, whereas NNDTC was computed using HALO software. Subsequently, we analyzed the association of these parameters with patient outcomes (progression-free survival [PFS] and overall survival [OS]), clinicopathologic features, and denosumab treatment responsiveness. RESULTS: Low LIS was indicative of both poor PFS and OS (both P < .001). In addition, LIS was significantly associated with sex ( P = .046), Enneking staging ( P < .001), Ki-67 expression ( P = .007), and denosumab treatment responsiveness ( P = .005). Lower CD8⁺ (tumor interior [TI]) NNDTC, and CD3⁺ (TI) NNDTC were associated with worse PFS ( P = .003 and .038, respectively), whereas lower CD8⁺ (TI) NNDTC was associated with worse OS ( P = .001), but CD8⁺ (tumor infiltrating margin) NNDTC had the opposite effect ( P = .002). Moreover, NNDTC showed a correlation with several clinicopathologic features. Importantly, LIS outperformed Enneking and Campanacci staging systems in predicting the clinical outcomes of GCTB. CONCLUSION: These findings suggest that LIS is a reliable predictive tool for clinically relevant outcomes and response to denosumab therapy in patients with GCTB. These parameters may prove to be useful in guiding prognostic risk stratification and therapeutic optimization for patients.

Background: Currently, there is limited understanding regarding the clinical significance of the tumor-stroma ratio (TSR) in giant cell tumor of bone (GCTB). Hence, we aimed to investigate the distribution of TSR in GCTB and explore its correlation with various clinicopathologic factors, immune microenvironment, survival prognosis, and denosumab treatment responsiveness. Methods: We conducted a multicenter cohort study comprising 426 GCTB patients treated at four centers. TSR was evaluated on hematoxylin and eosin-stained and immunofluorescent sections of tumor specimens. Immunohistochemistry was performed to assess CD3+, CD4+, CD8+, CD20+, PD-1+, PD-L1+, and FoxP3+ TIL subtypes as well as Ki-67 expression levels in 426 tissue specimens. These parameters were then analyzed for their correlations with patient outcomes [local recurrence-free survival (LRFS) and overall survival (OS)], clinicopathological features, and denosumab treatment responsiveness. Results: Low TSR was significantly associated with poor LRFS and OS in both cohorts. Furthermore, TSR was also correlated with multiple clinicopathological features, TIL subtype expression, and denosumab treatment responsiveness. TSR demonstrated similar predictive capabilities as the conventional Campanacci staging system for predicting patients' LRFS and OS. Conclusion: The results of this study provide evidence supporting the use of TSR as a reliable prognostic tool in GCTB and as a predictor of denosumab treatment responsiveness. These findings may aid in developing individualized treatment strategies for GCTB patients in the future.