Spatial expression of fibroblast activation protein-α in clear cell renal cell carcinomas revealed by multiplex immunoprofiling analysis of the tumor microenvironment.

Clear cell renal cell carcinoma (ccRCC) is one of the most challenging neoplasms because of its phenotypic variability and intratumoral heterogeneity. Because of its variability, ccRCC is a good test bench for the application of new technological approaches to unveiling its intricacies. Multiplex immunofluorescence (mIF) is an emerging method that enables the simultaneous and detailed assessment of tumor and stromal cell subpopulations in a single tissue section. This novel approach represents a promising step forward for analyzing the microenvironmental cell composition and distribution across the tumor and understanding its possible interactions with tumor cells. This study provides the first characterization of the spatial distribution of fibroblast activation protein-α (FAP)-expressing cancer-associated fibroblasts (FAP + CAFs) in conjunction with lymphoid (CD4 + , CD8 + , CD4 + FOXP3 + , and CD20 +) and myeloid (CD68 +) cells in tissue sections from ccRCC in their early phases of evolution (n = 88). Both the tumor center and periphery were analyzed with mIF. FAP + CAFs and tumor-infiltrating lymphocytes (TILs) were significantly concentrated at the tumor periphery. Additionally, elevated percentages of FAP + CAFs were correlated with larger tumors and synchronous metastases. Increased levels of CD68 +  and CD4 + FOXP3 +  cells (above the 75th percentile) were linked to worse cancer-specific survival (CSS) in patients with ccRCC. Furthermore, significant correlations emerged among FAP + CAFs, TILs, and CD68 +  cells, and the co-occurrence of elevated FAP + CAFs, T-cytotoxic (CD8 +), T-regulatory (CD4 + FOXP3 +) cells, and macrophages (CD68 +) at the tumor center were independently associated with worse CSS. These findings suggest that FAP + CAFs contribute to the aggressiveness of ccRCC, and their role is potentially mediated by their ability to foster an immunosuppressive environment within the renal tumor microenvironment.

Larrinaga G ，Redrado M ，Loizaga-Iriarte A ，Pérez-Fernández A ，Santos-Martín A ，Angulo JC ，Fernández JA ，Calvo A ，López JI ... -  《-》

Fibroblast Activation Protein-α Expression in Cancer-Associated Fibroblasts Shows the Poor Survival of Colorectal Cancer via Immune-Mediated Pathways : Implications of FAP in Cancer-Associated Fibroblasts Link Immune Dysregulation to Adverse Survival in C

Cancer-associated fibroblasts (CAFs) and immune cells, the key components of the tumor microenvironment (TME), play critical roles in oncogenesis. Despite the recognized function of fibroblast activation protein-α (FAP), a specific biomarker of CAFs in cancer progression, its role in the survival of patients with colorectal cancer (CRC) and tumor immune microenvironment (TIME) remains unclear. We investigated 180 pathological sections obtained from 178 consecutive patients with CRC who underwent surgical resection at Shiga University of Medical Science Hospital between January 2013 and December 2015. FAP expression levels and CD3 and CD8 densities at the invasive margin and center of tumor were assessed using immunohistochemical (IHC) staining. Furthermore, we used single-cell RNA sequencing (scRNA-seq) of CAFs in a separate cohort of 10 untreated patients with CRC derived from the Gene Expression Omnibus database. According to IHC evaluation, high FAP expression in patients with CRC showed a correlation with reduced tumor-infiltrating lymphocyte (TIL) distribution and poor survival. Based on the FAP transcription levels obtained through scRNA-seq analysis, CAFs were grouped into high and low FAP expression groups. Elevated FAP expression was correlated with decreased expression of T- and B-cell biomarkers, suggesting an association with an immunosuppressive TME promotion. Several genes associated with cancer-related immune-mediated pathways (CXCL12, COL11A1, CCL11, and COL10A1) were significantly upregulated in FAP-positive CAFs. This study highlights the effects of FAP expression on survival of patients with CRC, its interaction with TILs, and relevant signaling pathways, and underscores potential immunotherapeutic targets for future investigation.

Qin Y ，Miyake T ，Muramoto K ，Maekawa T ，Nishina Y ，Wang Y ，Shimizu T ，Tani M ... -  《-》

Prognostic Value of Cancer-Associated Fibroblast Marker Expression in the Intratumoral and Marginal Areas of Soft Tissue Sarcoma.

The tumor microenvironment of sarcomas has not been studied in detail; in particular, little is known about cancer-associated fibroblasts (CAFs). Sarcoma cells are difficult to distinguish from CAFs, either histomorphologically or immunohistochemically. We scored the expression of individual CAF markers (fibroblast-activating protein [FAP], CD10, and podoplanin) in the intratumoral and marginal areas of 133 sarcomas. We also examined the association between these markers, as well as the number of CD163-positive macrophages (i.e., tumor-associated macrophages), and clinical outcome. In all cases, the log-rank test revealed that those with high marker scores and macrophage counts (except for marginal CD10+ CAFs) showed significantly worse disease-free survival (DFS). Grade 2/3 cases with high CAF scores (excluding the marginal FAP and CD10 scores) showed significantly worse DFS, whereas those with high intratumoral FAP/CD10 and marginal podoplanin scores showed significantly worse metastasis-free survival (MFS), and those with high intratumoral CD10 score showed significantly worse local recurrence-free survival (LFS). Multivariate analysis identified intratumoral CD10/podoplanin scores and marginal FAP/podoplanin scores as independent prognostic factors for DFS, intratumoral FAP/CD10 and marginal FAP/podoplanin/CD163-positive macrophage scores as independent prognostic factors for MFS, and the intratumoral podoplanin score as an independent prognostic factor for LFS. There was a weak-to-moderate correlation between each score and CD163-positive macrophage counts. Patients with high CAF marker expression in the intratumoral and marginal areas have a poorer outcome. The tumor microenvironment of sarcomas has not been studied in detail; in particular, little is known about cancer-associated fibroblasts (CAFs). Sarcoma cells are difficult to distinguish from CAFs, either histomorphologically or immunohistochemically. We scored the expression of individual CAF markers (fibroblast-activating protein [FAP], CD10, and podoplanin) in the intratumoral and marginal areas of 133 sarcomas. We also examined the association between these markers, as well as the number of CD163-positive macrophages (i.e., tumor-associated macrophages), and clinical outcome. In all cases, the log-rank test revealed that those with high marker scores and macrophage counts (except for marginal CD10+ CAFs) showed significantly worse disease-free survival (DFS). Grade 2/3 cases with high CAF scores (excluding the marginal FAP and CD10 scores) showed significantly worse DFS, whereas those with high intratumoral FAP/CD10 and marginal podoplanin scores showed significantly worse metastasis-free survival (MFS), and those with high intratumoral CD10 score showed significantly worse local recurrence-free survival (LFS). Multivariate analysis identified intratumoral CD10/podoplanin scores and marginal FAP/podoplanin scores as independent prognostic factors for DFS, intratumoral FAP/CD10 and marginal FAP/podoplanin/CD163-positive macrophage scores as independent prognostic factors for MFS, and the intratumoral podoplanin score as an independent prognostic factor for LFS. There was a weak-to-moderate correlation between each score and CD163-positive macrophage counts. Patients with high CAF marker expression in the intratumoral and marginal areas have a poorer outcome.

Umakoshi M ，Kudo-Asabe Y ，Tsuchie H ，Li Z ，Koyama K ，Miyabe K ，Yoshida M ，Nagasawa H ，Nanjo H ，Okada K ，Maeda D ，Miyakoshi N ，Tanaka M ，Goto A ... -  《-》

Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response. The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.

Gallant JP ，Hintz HM ，Gunaratne GS ，Breneman MT ，Recchia EE ，West JL ，Ott KL ，Heninger E ，Jackson AE ，Luo NY ，Rosenkrans ZT ，Hernandez R ，Zhao SG ，Lang JM ，Meimetis L ，Kosoff D ，LeBeau AM ... -  《-》

Deciphering potential molecular mechanisms in clear cell renal cell carcinoma based on the ubiquitin-conjugating enzyme E2 related genes: Identifying UBE2C correlates to infiltration of regulatory T cells.

Renal clear cell carcinoma (ccRCC) is a highly aggressive and common form of kidney cancer, with limited treatment options for advanced stages. Recent studies have highlighted the importance of the ubiquitin-proteasome system in tumor progression, particularly the role of ubiquitin-conjugating enzyme E2 (UBE2) family members. However, the prognostic significance of UBE2-related genes (UBE2RGs) in ccRCC remains unclear. In this study, bulk RNA-sequencing and single-cell RNA-sequencing data from ccRCC patients were retrieved from the Cancer Genome Atlas and Gene Expression Omnibus databases. Differential expression analysis was performed to identify UBE2RGs associated with ccRCC. A combination of 10 machine learning methods was applied to develop an optimal prognostic model, and its predictive performance was evaluated using area under the curve (AUC) values for 1-, 3-, and 5-year overall survival (OS) in both training and validation cohorts. Functional enrichment analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes were conducted to explore the biological pathways involved. Correlation analysis was conducted to investigate the association between the risk score and tumor mutational burden (TMB) and immune cell infiltration. Immunotherapy and chemotherapy sensitivity were assessed by immunophenoscore and tumor immune, dysfunction, and exclusion scores to identify potential predictive significance. In vitro, knockdown of the key gene UBE2C in 786-O cells by specific small interfering RNA to validate its impact on apoptosis, migration, cell cycle, migration, invasion of tumor cells, and induction of regulatory T cells (Tregs). Analysis of sc-RNA revealed that UBE2 activity was significantly upregulated in malignant cells, suggesting its role in tumor progression. A three-gene prognostic model comprising UBE2C, UBE2D3, and UBE2T was constructed by Lasoo Cox regression and demonstrated robust predictive accuracy, with AUC values of 0.745, 0.766, and 0.771 for 1-, 3-, and 5-year survival, respectively. The model was validated as an independent prognostic factor in ccRCC. Patients in the high-risk group had a worse prognosis, higher TMB scores, and low responsiveness to immunotherapy. Additionally, immune infiltration and chemotherapy sensitivity analyses revealed that UBE2RGs are associated with various immune cells and drugs, suggesting that UBE2RGs could be a potential therapeutic target for ccRCC. In vitro experiments confirmed that the reduction of UBE2C led to an increase in apoptosis rate, as well as a decrease in tumor cell invasion and metastasis abilities. Additionally, si-UBE2C cells reduced the release of the cytokine Transforming Growth Factor-beta 1 (TGF-β1), leading to a decreased ratio of Tregs in the co-culture system. This study presents a novel three-gene prognostic model based on UBE2RGs that demonstrates significant predictive value for OS, immunotherapy, and chemotherapy in ccRCC patients. The findings underscore the potential of UBE2 family members as biomarkers and therapeutic targets in ccRCC, warranting further investigation in prospective clinical trials.

Feng X ，Wang Z ，Cen M ，Zheng Z ，Wang B ，Zhao Z ，Zhong Z ，Zou Y ，Lv Q ，Li S ，Huang L ，Huang H ，Qiu X ... -  《-》