Endosialin-positive CAFs promote hepatocellular carcinoma progression by suppressing CD8+ T cell infiltration.

BACKGROUND AND AIMS: Endosialin, also known as tumor endothelial marker1 or CD248, is a transmembrane glycoprotein that is mainly expressed in cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC). Our previous study has found that endosialin-positive CAFs could recruit and induce the M2 polarization of macrophages in HCC. However, whether they may regulate other types of immune cells to promoting HCC progression is not known. APPROACH AND RESULTS: The growth of both subcutaneous and orthotopic HCC tumors was significantly inhibited in endosialin knockout (ENKO) mice. Single-cell sequencing and flow cytometry analysis showed that tumor tissues from ENKO mice had increased CD8+ T cell infiltration. Mixed HCC tumor with Hepa1-6 cells and endosialin knockdown fibroblasts also showed inhibited growth and increased CD8+ T cell infiltration. Data from in vitro co-culture assay, chemokine array and antibody blocking assay, RNA-seq and validation experiments showed that endosialin inhibits the phosphorylation and nuclear translocation of STAT1 in CAFs. This inhibition leads to a decrease in CXCL9/10 expression and secretion, resulting in the suppression of CD8+ T cell infiltration. High level of endosialin protein expression was correlated with low CD8+ T infiltration in the tumor tissue of HCC patients. The combination therapy of endosialin antibody and PD-1 antibody showed synergistic antitumor effect compared with either antibody used individually. CONCLUSIONS: Endosialin could inhibit CD8+ T cell infiltration by inhibiting the expression and secretion of CXCL9/10 in CAFs, thus promote HCC progression. Combination therapy with endosialin antibody could increase the antitumor effect of PD-1 antibody in HCC, which may overcome the resistance to PD-1 blockade.

The role of phase separation in RNA modification: both cause and effect.

Phase separation is a critical mechanism for partitioning cellular functions by specific aggregation of biological macromolecules. Recent studies have found that phase separation is widely contributed in various biological functions, particularly in RNA related processes. Over 170 different post-transcriptional modifications occur in RNA, which is considered to be one of the most important physiological and pathogenic epigenetic mechanisms. Here, we discuss the role of phase separation in regulating RNA modification processing to ensure orderly RNA metabolism and function. Enzymes responsible for RNA modification undergo compartmentalization, enabling them to traffic client RNAs and amplify modifying efficacy. Meanwhile, altered RNA affects the formation, dissolution, and biophysical properties of phase separation conversely. These findings deeper our understanding of the interplay between phase separation and RNAs that governs a wide range of cellular processes. Finally, we concluded pathological roles of phase separation in RNA modification towards clinical applications and outlined perspectives to research RNA modification through the lens of phase separation.

YAP1 Inhibition Induces Phenotype Switching of Cancer-Associated Fibroblasts to Tumor Suppressive in Prostate Cancer.

Prostate cancer (PCa) rarely responds to immune-checkpoint blockade (ICB) therapies. Cancer-associated fibroblasts (CAFs) are critical components of the immunologically "cold" tumor microenvironment and are considered a promising target to enhance the immunotherapy response. In this study, we aimed to reveal the mechanisms regulating CAF plasticity to identify potential strategies to switch CAFs from pro-tumorigenic to anti-tumor phenotypes and enhance ICB efficacy in PCa. Integration of four PCa single-cell RNA-sequencing datasets defined pro-tumorigenic and anti-tumor CAFs, and RNA-seq, flow cytometry, and a PCa organoid model demonstrated the functions of two CAF subtypes. Extracellular matrix-associated CAFs (ECM-CAF) promoted collagen deposition and cancer cell progression, and lymphocyte-associated CAFs (Lym-CAF) exhibited an anti-tumor phenotype and induced the infiltration and activation of CD8+ T cells. YAP1 activity regulated the ECM-CAF phenotype, and YAP1 silencing promoted switching to Lym-CAFs. NF-κB p65 was the core transcription factor in the Lym-CAF subset, and YAP1 inhibited nuclear translocation of p65. Selective depletion of YAP1 in ECM-CAFs in vivo promoted CD8+ T-cell infiltration and activation and enhanced the therapeutic effects of anti- PD-1 treatment in PCa. Overall, this study revealed a mechanism regulating CAF identity in PCa and highlighted a therapeutic strategy for altering the CAF subtype to suppress tumor growth and increase sensitivity to ICB.

Endosialin in Cancer: Expression Patterns, Mechanistic Insights, and Therapeutic Approaches.

Endosialin, also known as tumor endothelial marker 1 (TEM1) or CD248, is a single transmembrane glycoprotein with a C-type lectin-like domain. Endosialin is mainly expressed in the stroma, especially in cancer-associated fibroblasts and pericytes, in most solid tumors. Endosialin is also expressed in tumor cells of most sarcomas. Endosialin can promote tumor progression through different mechanisms, such as promoting tumor cell proliferation, adhesion and migration, stimulating tumor angiogenesis, and inducing an immunosuppressive tumor microenvironment. Thus, it is considered an ideal target for cancer treatment. Several endosialin-targeted antibodies and therapeutic strategies have been developed and have shown preliminary antitumor effects. Here, we reviewed the endosialin expression pattern in different cancer types, discussed the mechanisms by which endosialin promotes tumor progression, and summarized current therapeutic strategies targeting endosialin.

CD248 promotes migration and metastasis of osteosarcoma through ITGB1-mediated FAK-paxillin pathway activation.

BACKGROUND: Osteosarcoma (OS) is the most common malignant bone tumor with a high incidence in children and adolescents. Frequent tumor metastasis and high postoperative recurrence are the most common challenges in OS. However, detailed mechanism is largely unknown. METHODS: We examined the expression of CD248 in OS tissue microarrays by immunohistochemistry (IHC) staining. We studied the biological function of CD248 in cell proliferation, invasion and migration of OS cells by CCK8 assay, transwell and wound healing assay. We also studied its function in the metastasis of OS in vivo. At last, we explored the potential mechanism how CD248 promotes OS metastasis by using RNA-seq, western blot, immunofluorescence staining and co-immunoprecipitation using CD248 knockdown OS cells. RESULTS: CD248 was highly expressed in OS tissues and its high expression was correlated with pulmonary metastasis of OS. Knockdown of CD248 in OS cells significantly inhibited cell migration, invasion and metastasis, while had no obvious effect on cell proliferation. Lung metastasis in nude mice was significantly inhibited when CD248 was knocked down. Mechanistically, we found that CD248 could promote the interaction between ITGB1 and extracellular matrix (ECM) proteins like CYR61 and FN, which activated the FAK-paxillin pathway to promote the formation of focal adhesion and metastasis of OS. CONCLUSION: Our data showed that high CD248 expression is correlated with the metastatic potential of OS. CD248 may promote migration and metastasis through enhancing the interaction between ITGB1 and certain ECM proteins. Therefore, CD248 is a potential marker for diagnosis and effective target for the treatment of metastatic OS.

Single-cell analysis reveals the COL11A1+ fibroblasts are cancer-specific fibroblasts that promote tumor progression.

Background: Cancer-associated fibroblasts (CAFs) promote tumor progression through extracellular matrix (ECM) remodeling and extensive communication with other cells in tumor microenvironment. However, most CAF-targeting strategies failed in clinical trials due to the heterogeneity of CAFs. Hence, we aimed to identify the cluster of tumor-promoting CAFs, elucidate their function and determine their specific membrane markers to ensure precise targeting. Methods: We integrated multiple single-cell RNA sequencing (scRNA-seq) datasets across different tumors and adjacent normal tissues to identify the tumor-promoting CAF cluster. We analyzed the origin of these CAFs by pseudotime analysis, and tried to elucidate the function of these CAFs by gene regulatory network analysis and cell-cell communication analysis. We also performed cell-type deconvolution analysis to examine the association between the proportion of these CAFs and patients' prognosis in TCGA cancer cohorts, and validated that through IHC staining in clinical tumor tissues. In addition, we analyzed the membrane molecules in different fibroblast clusters, trying to identify the membrane molecules that were specifically expressed on these CAFs. Results: We found that COL11A1+ fibroblasts specifically exist in tumor tissues but not in normal tissues and named them cancer-specific fibroblasts (CSFs). We revealed that these CSFs were transformed from normal fibroblasts. CSFs represented a more activated CAF cluster and may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. High CSF proportion was associated with poor prognosis in bladder cancer (BCa) and lung adenocarcinoma (LUAD), and IHC staining of COL11A1 confirmed their specific expression in tumor stroma in clinical BCa samples. We also identified that CSFs specifically express the membrane molecules LRRC15, ITGA11, SPHK1 and FAP, which could distinguish CSFs from other fibroblasts. Conclusion: We identified that CSFs is a tumor specific cluster of fibroblasts, which are in active state, may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. Membrane molecules LRRC15, ITGA11, SPHK1 and FAP could be used as therapeutic targets for CSF-targeting cancer treatment.

Single-cell analysis of multiple cancer types reveals differences in endothelial cells between tumors and normal tissues.

Endothelial cells (ECs) play an important role in tumor progression. Currently, the main target of anti-angiogenic therapy is the vascular endothelial growth factor (VEGF) pathway. Some patients do benefit from anti-VEGF/VEGFR therapy; however, a large number of patients do not have response or acquire drug resistance after treatment. Moreover, anti-VEGF/VEGFR therapy may lead to nephrotoxicity and cardiovascular-related side effects due to its action on normal ECs. Therefore, it is necessary to identify targets that are specific to tumor ECs and could be applied to various cancer types. We integrated single-cell RNA sequencing data from six cancer types and constructed a multi-cancer EC atlas to decode the characteristic of tumor ECs. We found that tip-like ECs mainly exist in tumor tissues but barely exist in normal tissues. Tip-like ECs are involved in the promotion of tumor angiogenesis and inhibition on anti-tumor immune responses. Moreover, tumor cells, myeloid cells, and pericytes are the main sources of pro-angiogenic factors. High proportion of tip-like ECs is associated with poor prognosis in multiple cancer types. We also identified that prostate-specific membrane antigen (PSMA) is a specific marker for tip-like ECs in all the cancer types we studied. In summary, we demonstrate that tip-like ECs are the main differential EC subcluster between tumors and normal tissues. Tip-like ECs may promote tumor progression through promoting angiogenesis while inhibiting anti-tumor immune responses. PSMA was a specific marker for tip-like ECs, which could be used as a potential target for the diagnosis and treatment of non-prostate cancers.

Endosialin-positive tumor-derived pericytes promote tumor progression through impeding the infiltration of CD8+ T cells in clear cell renal cell carcinoma.

BACKGROUND: Immune checkpoint blockade (ICB) therapy can be effective against clear cell renal cell carcinoma (ccRCC), but many patients show no benefit. Tumor-derived pericytes (TDPs) may promote tumor progression by influencing T cells and are an immunotherapy target; however, they may comprise functionally distinct subtypes. We aimed to identify markers of tumor-promoting TDPs and develop TDP-targeting strategies to enhance ICB therapy effectiveness against ccRCC. METHODS: We analyzed the relationship between endosialin (EN) expression and cytotoxic T-lymphocyte (CTL) infiltration in ccRCC tumor samples using flow cytometry and in a ccRCC-bearing mice inhibited for EN via knockout or antibody-mediated blockade. The function of ENhigh TDPs in CTL infiltration and tumor progression was analyzed using RNA-sequencing (RNA-seq) data from ccRCC tissue-derived TDPs and single-cell RNA-seq (scRNA-seq) data from an online database. The role of EN in TDP proliferation and migration and in CTL infiltration was examined in vitro. Finally, we examined the anti-tumor effect of combined anti-EN and anti-programmed death 1 (PD-1) antibodies in ccRCC-bearing mice. RESULTS: High EN expression was associated with low CTL infiltration in ccRCC tissues, and inhibition of EN significantly increased CTL infiltration in ccRCC-bearing mice. RNA-seq and scRNA-seq analyses indicated that high EN expression represented the TDP activation state. EN promoted TDP proliferation and migration and impeded CTL infiltration in vitro. Finally, combined treatment with anti-EN and anti-PD-1 antibodies synergistically enhanced anti-tumor efficacy. CONCLUSION: ENhigh TDPs are in an activated state and inhibit CTL infiltration into ccRCC tissues. Combined treatment with anti-EN and anti-PD-1 antibodies may improve ICB therapy effectiveness against ccRCC.