Differences in tumor-associated cytokine production and immune mechanisms between myofibroblastic cancer associated fibroblasts and myofibroblasts.

Patients with multifocal, locally advanced prostate cancer are at the highest risk of recurrence and death. Neoadjuvant therapies have limited response rates in this patient cohort and there remains a critical need to develop curative treatments. Prostate cancer (PC) shows marked heterogeneity, which is not limited to tumor cells but extends across immune and stromal compartments and has been linked to metastatic disease and therapeutic resistance. Cancer associated fibroblasts (CAFs) have been proposed to play a critical role in PC progression and invasion, but it is still unclear how CAFs interact with tumor cells. We evaluated CAFs from prostatectomy specimens as drivers of resistance to neoadjuvant androgen receptor signaling inhibitor (ARSi) and chemotherapy and assessed treatment response in co-culture systems in the context of CAF presence. We have utilized a novel radio-pathology tool that integrates PSMA PET/MRI scans to identify regions of interest that associate with heterogenous treatment response. MRI scans were utilized to print patient-specific 3D molds to micro dissect live tissue for subsequent cell sorting and molecular analyses. We have established a multi-parameter flow cytometry panel to characterize primary prostate fibroblasts from tumor foci and adjacent normal prostate tissue. In addition, co-culture assays were performed including immortalized CAF (hPrCSC-44) and MSC-derived fibroblast. To examine the tumor-promoting role of CAFs, fibroblasts were co-cultured with androgen-sensitive 3D tumor PC spheroids (LNCaP, C42B and LAPC4) in the integrated microfluidic STACKs platform that allows for the assessment of spatio-temporal interaction in a variety of culture microenvironments to model complex interactions. Cytotoxicity in 3D PC spheroids was assessed after Apalutamide, Darolutamide, or Docetaxel treatment by confocal microscopy. Integrated DNA, RNA, and radiology analysis of tumor biopsies have identified gene signatures associated with early biochemical recurrence. Most biopsies had low tumor purity, evidence of the cytotoxic impact of chemohormonal therapy while high tumor purity predicted high tumor expression. Poor clinical outcome was significantly associated with elevated stromal scores while epithelial and stromal enrichment were inversely correlated (rho= -0.80, p< 5 × 10−16). The presence of CAFs significantly decreased Docetaxel (C42B only vs C42B+CAF, 86.07% vs. 44.57%, respectively, p<0.001; LAPC4 only vs LAPC4+CAF, 70.98% vs 26.64%, respectively, p<0.001) and Darolutamide-induced (C42B only vs C42B+CAF, 52.59% vs 37.87%, respectively, p=0.0407). In conclusion, recurrent PC is linked to increased stromal content and the presence of CAFs supported tumor survival in response to ARSi and Docetaxel treatment. The molecular drivers of this phenomenon are currently being investigated. Citation Format: Nikolett Lupsa, Erika Heninger, Adeline Ding, Shannon R. Reese, Xavier T. Hazelberg, Aaron M. LeBeau, Brian P. Johnson, Peter P. Geiger, David J. Beebe, David A. Quigley, Joshua M. Lang. Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 298.

Cyclosporine A (CsA) is a widely used immunosuppressant, with clinical applications ranging from organ transplants to chronic inflammatory diseases. One of the side effects associated with CsA treatment is the development of gingival overgrowth. Exuberant growth of connective tissue within the periodontium can result from hyperactivity of resident fibroblasts. Fibroblasts are capable of secreting interleukin-6 (IL-6), which has been shown to enhance proliferation as well as collagen and glycosaminoglycan synthesis by these cells. We tested the hypothesis that one of the pathogenetic mechanisms underlying CsA-induced fibrosis is an enhanced IL-6 secretion by gingival fibroblasts (GF) in response to this drug. The ability of CsA to upregulate GF IL-6 secretion alone or in combination with bacterial challenge or other inflammatory cytokines was tested in an in vitro system. Fibroblast cultures were established from systemically healthy gingival tissue donors and were challenged with CsA in the absence or presence of bacteria, IL-1beta, or tumor necrosis factor (TNF) alpha as co-stimulants. Nifedipine and phenytoin were also tested to further support findings with CsA. After 72 hours of incubation, culture supernatants were collected and analyzed for IL-6 content by ELISA. We have shown that GF respond to CsA with an increase in IL-6 secretion. The magnitude of this response varies among cultures derived from different tissue donors. We have also demonstrated that GF IL-6 responses to bacterial challenge or TNFalpha are downregulated by CsA. However, CsA synergizes with IL-1beta to further upregulate IL-6 secretion, and this effect is shared by phenytoin and nifedipine. We conclude that one of the pathogenetic mechanisms underlying drug-induced gingival overgrowth may be enhanced secretion of IL-6 by GF in response to these medications. This is the first report on direct and indirect effects of gingival overgrowth-related medications on GF IL-6 metabolism. This work will lay the foundation for future studies directed towards the development of prevention or treatment modalities for gingival overgrowth based on blocking the fibrogenic activities of IL-6 at the cellular level.

Soft gingiva is often compromised in gingival health; however, the underlying biological mechanisms remain unknown. Extracellular matrix (ECM) stiffness is involved in the progression of various fibroblast-related inflammatory disorders via cellular mechanotransduction. Gingival stiffness might regulate cellular mechanotransduction-mediated proinflammatory responses in gingival fibroblasts. This in vitro study aims to investigate the effects of substrate stiffness on proinflammatory responses in human gingival fibroblasts (hGFs). The hGFs isolated from two healthy donors cultured on type I collagen-coated polydimethylsiloxane substrates with different stiffnesses, representing soft (5 kPa) or hard (25 kPa) gingiva. Expression levels of proinflammatory mediators, prostaglandin E2 or interleukin-1β, in hGFs were significantly higher with the soft substrate than with the hard substrate, even without and with lipopolysaccharide (LPS) to induce inflammation. Expression levels of gingival ECM and collagen cross-linking agents in hGFs were downregulated more with the soft substrate than with the hard substrate through 14 days of culture. The soft substrate suppressed the expression of mechanotransduction-related transcriptional factors and activated the expression of inflammation-related factors, whereas the hard substrate demonstrated the opposite effects. Soft substrate induced proinflammatory responses and inhibition of ECM synthesis in hGFs by inactivating cellular mechanotransduction. This supports the importance of ECM stiffness in gingival health.

Acute Lymphoblastic Leukaemia (ALL) Cells and Commonly-Used Chemotherapy Drugs Can Induce Bone Marrow-Derived Mesenchymal Stromal Cells to Become Cancer Associated Fibroblasts (CAF), Leading to Enhanced ALL Proliferation and Treatment Resistance

In the tumor microenvironment, cancer-associated fibroblasts (CAFs) are known to play key roles in promoting cancer cell invasion and cancer progression. CAFs are also highly invasive compared with normal fibroblasts in a 3-D co-culture system with lung cancer cells. Due to CAF heterogeneity, there are various differences in cell motility even within a CAF population. To classify CAFs according to their motility, we named less motile CAFs as “static CAFs” and more motile CAFs as “motile CAFs”. To separate and isolate these static CAFs and motile CAFs from a 3-D co-culture system, CAFs were transfected with a green-to-red photoconvertible fluorescent protein, Dendra2. Dendra2-transfected CAFs at specific areas (i.e., static or motile CAFs) were UV-irradiated using a confocal microscope. CAFs with red fluorescence were then isolated by flow cytometry and were subjected to RNA sequencing. In gene ontology and gene set enrichment analyses, two types of CAFs showed significant differences in global gene expression patterns. These data suggest that heterogeneous CAFs with different motility have distinct roles in the regulation of various signaling pathways in the tumor microenvironment. Citation Format: Sieun Lee, Young-Ho Ahn. Microscope-based sorting of highly motile cancer-associated fibroblasts using a photoconvertible fluorescent protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3861.

Ovarian cancer is the most lethal gynecologic malignancy in the US. While major research efforts have been dedicated to identify disease related genes and mutations in cancer cells, the role of the stromal tumor microenvironment in disease progression is relatively unexplored. We previously reported that while ovarian cancer cells have minimal response to TGF-beta, TGF-beta induced secretory proteins from cancer associated fibroblasts (CAFs) promote ovarian cancer motility and invasion potential. The present study seeks to further evaluate the involvement of TGF-beta signaling in CAFs in the progression of ovarian cancer and the therapeutic potential of targeting such signaling in cancer treatment. By comparing the gene expression profiles of laser-microdissected ovarian tumor stromal tissues from high-grade serous ovarian cancer (HGSOC) patients with short and long survival, a differentially expressed gene signature was identified. Using the IPA platform, signaling pathways and upstream regulators activated in CAFs from patients with short survival were identified. TGF-beta signaling was found to be significantly activated (p = 2.47E-03), while the ligand TGF-beta 1 was identified as the most significant upstream regulator (Z-score = 5.189, p = 8.85E-29), suggested the involvement of CAF TGF-beta signaling in poor patient survival. Further immunohistochemistry and survival analyses revealed the clinical relevance of multiple TGF-beta regulated stromal genes, including VCAN, MFAP5 and COMP in poor disease prognosis. Subsequently, cell culture studies were performed to evaluate the effects of TGF-beta in ovarian cancer progression. While exogenous TGF-beta did not affect proliferation, motility and invasion potential of ovarian cancer cells directly, TGF-beta promoted ovarian cancer cells motility and invasion potential in a co-culture system of cancer cells and CAFs. Similarly, treatment of exogenous TGF-beta-induced CAF secretory proteins promoted ovarian cancer progression, suggested the presence of CAFs is required for TGF-beta mediated ovarian cancer progression and targeting such TGF-beta signaling in CAFs could be a novel modality of ovarian cancer treatment. To reprogram CAFs by targeting its TGF-beta signaling, multiple approaches were tested in co-culture systems. Using either siRNAs or small molecule inhibitors, TGF-beta signaling in CAFs was effectively suppressed and inhibited ovarian cancer progression was observed. Based on our current findings, animal studies will be performed to further evaluate the efficacy of targeting the CAF TGF-beta signaling pathway in ovarian cancer treatment. This study presents the paradigm which activation of TGF-beta signaling in ovarian CAFs is significantly associated to the poor clinical outcomes. Targeting such pathway activation in CAFs could effectively suppress tumor progression and thereby presented a novel approach in cancer treatment. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Reprogramming the TGF-beta signaling in cancer-associated fibroblasts inhibits ovarian cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2015-5066

Cancer-associated fibroblasts (CAFs), the predominant stromal cells in the tumor microenvironment (TME), play a critical role in the progression of solid tumors, including oral squamous cell carcinoma (OSCC). However, the molecular mechanisms by which OSCC-derived factors mediate CAF differentiation remain incompletely understood. This study investigates the role of the C-X-C motif chemokine ligand 1 (CXCL1), secreted by OSCC cells, in promoting CAF differentiation and its downstream impact on tumor progression. Gingival fibroblasts (GFs) were treated with conditioned medium (CM) from various OSCC cell lines to assess their potential to induce CAF differentiation. Proteomic analysis using liquid chromatography–mass spectrometry identified CXCL1 as a key factor highly secreted in SCC25-derived CM, which exhibited the strongest capacity to induce CAF differentiation. CXCL1 synergistically enhanced TGF-β1-induced differentiation of GFs into α-smooth muscle actin (αSMA)- and vimentin-expressing CAFs by approximately 1.5-fold, confirming its co-stimulatory function. Conversely, silencing its receptor CXCR2 reduced CAF marker expression by over 50%, indicating a strong inhibitory effect on CAF differentiation. In vivo, co-injection of SCC25 cells with GFs significantly promoted tumor growth and stromal CAF marker expression, whereas CXCR2 knockdown in GFs led to a ~ 40% reduction in tumor volume and reduced αSMA/vimentin-positive CAFs. These findings establish CXCL1 as a pivotal mediator of CAF differentiation through CXCR2-dependent signaling, and highlight that the CXCL1–CXCR2 axis is a promising therapeutic target for modulating stromal–tumor interactions in OSCC.

A major cause of chronic inflammatory periodontal disease is Porphyromonas gingivalis, a non-motile, Gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known. The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli. The expression of immune response molecules was quantified by real-time polymerase chain reaction and enzyme-linked immunoassay. AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide, and monocyte chemotactic protein-1 expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1β, IL-6, and monocyte chemotactic protein-1, relative to stimulation by E. coli LPS. These findings demonstrate that E. coli LPS induces a stronger cytokine and chemokine response in gingival fibroblasts, whereas P. gingivalis LPS induces a stronger response in macrophages.

WGCNA reveals a biomarker for cancer-associated fibroblasts to predict prognosis in cervical cancer.

BackgroundProgrammed cell death (PCD) plays a crucial role in oral cancer pathogenesis and treatment. However, a comprehensive bibliometric analysis of the global research landscape in this field has not been conducted. This study aims to analyze the evolution and current trends of PCD research in oral cancer from 2000 to 2024.MethodsPublications were retrieved from the Web of Science Core Collection database using relevant keywords related to oral cancer and PCD. VOSviewer 1.6.20 and CiteSpace 6.1R6 software were employed to conduct bibliometric analysis, including publication trends, citation analysis, co-authorship networks, keyword co-occurrence, and research hotspots. The time span was set from January 2000 to December 2024.ResultsA total of 963 publications were identified and analyzed. The annual publication output showed a steady increase, with a significant growth rate after 2010, dividing the study period into three distinct phases. The most productive countries were China (58.42%), South Korea (12.27%), and Japan (10.04%), with China Medical University and Kaohsiung Medical University being the leading institutions. Research hotspots evolved from traditional apoptosis studies to emerging forms of PCD such as autophagy, ferroptosis, and pyroptosis. Keyword analysis revealed three major research clusters: basic molecular mechanisms (centered around ROS and oxidative stress), clinical aspects (including prognosis and cell proliferation), and cell death pathways. Citation burst analysis identified emerging trends in targeting multiple PCD pathways simultaneously for oral cancer therapy, with special focus on treatment resistance and survival.ConclusionThis bibliometric analysis provides a comprehensive overview of global research trends in PCD and oral cancer over the past two decades. The findings highlight the shift from basic mechanistic studies focusing on apoptosis to more diverse PCD pathways and translational research. Emerging research directions include the exploration of synergistic mechanisms among multiple PCD pathways, development of AI-based personalized treatment plans, investigation of microenvironment regulation of PCD, and application of novel drug delivery systems. These trends demonstrate the field's evolution toward more integrated, personalized approaches in oral cancer treatment. This study offers valuable insights for researchers and funding agencies to identify research gaps and potential collaboration opportunities in this rapidly developing field.

Periodontal disease is caused by dental plaque biofilms. Fusobacterium nucleatum is an important periodontal pathogen involved in the development of bacterial complexity in dental plaque biofilms. Human gingival fibroblasts (GFs) act as the first line of defense against oral microorganisms and locally orchestrate immune responses by triggering the production of reactive oxygen species and pro-inflammatory cytokines (IL-6 and IL-8). The frequency and severity of periodontal diseases is known to increase in elderly subjects. However, despite several studies exploring the effects of aging in periodontal disease, the underlying mechanisms through which aging affects the interaction between F. nucleatum and human GFs remain unclear. To identify genes affected by infection, aging, or both, we performed an RNA-Seq analysis using GFs isolated from a single healthy donor that were passaged for a short period of time (P4) ‘young GFs’ or for longer period of time (P22) ‘old GFs’, and infected or not with F. nucleatum. Comparing F. nucleatum-infected and uninfected GF(P4) cells the differentially expressed genes (DEGs) were involved in host defense mechanisms (i.e., immune responses and defense responses), whereas comparing F. nucleatum-infected and uninfected GF(P22) cells the DEGs were involved in cell maintenance (i.e., TGF-β signaling, skeletal development). Most DEGs in F. nucleatum-infected GF(P22) cells were downregulated (85%) and were significantly associated with host defense responses such as inflammatory responses, when compared to the DEGs in F. nucleatum-infected GF(P4) cells. Five genes (GADD45b, KLF10, CSRNP1, ID1, and TM4SF1) were upregulated in response to F. nucleatum infection; however, this effect was only seen in GF(P22) cells. The genes identified here appear to interact with each other in a network associated with free radical scavenging, cell cycle, and cancer; therefore, they could be potential candidates involved in the aged GF’s response to F. nucleatum infection. Further studies are needed to confirm these observations.

PurposeThis evidence mapping aims to describe and assess the quality of available evidence in systematic reviews (SRs) on treatments for oral cancer.Materials and methodsWe followed the methodology of Global Evidence Mapping. Searches in MEDLINE, EMBASE, Epistemonikos and The Cochrane Library were conducted to identify SRs on treatments for oral cancer. The methodological quality of SRs was assessed using the Assessing the Methodological Quality of Systematic Reviews-2 tool. We organized the results according to identified Population–Intervention–Comparison–Outcome (PICO) questions and presented the evidence mapping in tables and a bubble plot.ResultsFifteen SRs met the eligibility criteria, including 118 individual reports, of which 55.1% were randomized controlled clinical trials. Ten SRs scored “Critically low” methodological quality. We extracted 30 PICOs focusing on interventions such as surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy; 18 PICOs were for resectable oral cancer, of which 8 were reported as beneficial. There were 12 PICOs for unresectable oral cancer, of which only 2 interventions were reported as beneficial.ConclusionThere is limited available evidence on treatments for oral cancer. The methodological quality of most included SRs scored “Critically low”. The main beneficial treatment reported by authors for patients with resectable oral cancer is surgery alone or in combination with radiotherapy or chemotherapy. Evidence about the benefits of the treatments for unresectable oral cancer is lacking. These findings highlight the need to address future research focused on new treatments and knowledge gaps in this field, and increased efforts are required to improve the methodology quality and reporting process of SRs on treatments for oral cancer.

Cancer-associated fibroblasts (CAFs) play a substantial role in the tumor microenvironment, exhibiting a strong association with the advancement of various types of cancer, including breast, pancreatic, and prostate cancer. CAFs represent the most abundant mesenchymal cell population in breast cancer. Through diverse mechanisms, including the release of cytokines and exosomes, CAFs contribute to the progression of breast cancer by influencing tumor energy metabolism, promoting angiogenesis, impairing immune cell function, and remodeling the extracellular matrix. Moreover, CAFs considerably impact the response to treatment in breast cancer. Consequently, the development of interventions targeting CAFs has emerged as a promising therapeutic approach in the management of breast cancer. This article provides an analysis of the role of CAFs in breast cancer, specifically in relation to diagnosis, treatment, drug resistance, and prognosis. The paper succinctly outlines the diverse mechanisms through which CAFs contribute to the malignant behavior of breast cancer cells, including proliferation, invasion, metastasis, and drug resistance. Furthermore, the article emphasizes the potential of CAFs as valuable tools for early diagnosis, targeted therapy, treatment resistance, and prognosis assessment in breast cancer, thereby offering novel approaches for targeted therapy and overcoming treatment resistance in this disease.

Epigenetic modulation and repression of miR-200b by cancer-associated fibroblasts contribute to cancer invasion and peritoneal dissemination in gastric cancer.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1beta (IL-1beta) induced RIG-I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG-I expression induced by lipopolysaccharide (LPS) or double-stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG-I in the expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG-I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG-I induction by these stimuli was examined. The expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts transfected with RIG-I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG-I in concentration- and time-dependent manners. We also examined the localization of RIG-I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA-induced RIG-I expression. Introduction of RIG-I cDNA into gingival fibroblasts resulted in enhanced expression of IL-1beta, IL-6 and IL-8; moreover, overexpression of RIG-I stimulated with LPS or dsRNA synergistically increased expression of IL-1beta, IL-6 and IL-8. RIG-I may have important roles in the innate immune response in the regulation of IL-1beta, IL-6 and IL-8 expression in gingival fibroblasts in response to LPS and dsRNA.