Pan-Cancer Single-Cell Analysis Revealing the Heterogeneity of Cancer-Associated Fibroblasts in Skin Tumors.

Chapter 3 - Role of cancer-associated fibroblasts in tumor microenvironment

The interactions between neoplastic cells and their tumor microenvironment (TME) have become increasingly evident. Cancer-associated fibroblasts (CAFs) and M2 macrophages (M2 Mθ) are important components of the TME that have been recognized to orchestrate tumor-promoting inflammation and thereby tumor growth and progression. Here, we aim to determine the presence and role of CAFs and M2 Mθ in high grade cervical intraepithelial neoplasia (CIN 2/3) and invasive cervical carcinomas (SCCx). We optimized immunohistochemistry (IHC) markers for M2 Mθ (CD163, ARG1), CAFs (αSMA) and Th1 T cells (TBX21) in paraffin-embedded tissue sections from CIN2/3 (n=6) and SCCx cases (n=4). We found an increased infiltration of TBX21+ cells in the epithelium and stroma of CIN2/3 and SCCx cases compared to their normal adjacent stroma. This suggests that despite increased Th1 T cells, the progression from normal to CIN2/3 to SCCx might be facilitated by M2 Mθ and CAFs. Moreover, by performing double staining Immunofluorescence (IF) we found that M2 Mθ (CD163+) were the cells responsible for ARG1 production, an immunesuppressive mechanism utilized by these cells. We recently stained by IHC a cervical tissue microarray (TMA) of normal, CIN2/3 and SCCx cases (n=63) with CD163, αSMA and TBX21, and we are currently analyzing the correlation of these markers with clinicopathological data available for these cases (HPV status, tumor stage, therapy response, overall survival). With the hypothesis in mind that CAFs induce the polarization of macrophages into a M2 Mθ phenotype, we isolated fibroblasts from normal (n=2), CIN2/3 (n=1) and SCCx (n=2) fresh tissue explants obtained from surgery. We performed Immunocytochemistry (ICC) with αSMA, and observed a progressive increase in αSMA+ fibroblasts from normal to CIN2/3 and from CIN2/3 to SCCx fibroblasts, suggesting that CAFs might play an important role in the transformation of normal cervix into CIN2/3 and SCCx. Furthermore, we are currently performing co-culture experiments with normal, CIN2/3 and SCCx isolated fibroblasts and peripheral blood monocytes obtained from healthy donors. We will perform triple staining IF with vimentin, αSMA and CD163 in the slides generated from the co-culture, to evaluate if monocytes acquired a M2 Mθ phenotype with the presence of any of these 3 subsets of fibroblasts. Moreover, we will perform a cytokine analysis in the supernatants of these co-cultures to evaluate for potential cytokines produced by CAFs, implicated in M2 Mθ polarization. In this study we provide evidence regarding the role of CAFs in SCCx initiation and progression. Although the mechanisms that regulate fibroblast activation have not been elucidated, it is possible that CAFs might serve as novel therapeutic targets in cancer, either alone or in combination with existing anti-cancer therapies. Citation Format: Leonel Maldonado, Teresa Diaz-Montes, Abdulrahman Sinno, Edward J. Tanner, Christopher VandenBussche, Richard Roden, Iveta Yotova, Benjamin Tycko, Cornelia L. Trimble. Role and presence of cancer associated fibroblasts and M2 macrophages in high grade cervical intraepithelial neoplasia and invasive cervical carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3672. doi:10.1158/1538-7445.AM2014-3672

Background: Ecto-5'-nucleotidase (NT5E) encodes the cluster of differentiation 73 (CD73), whose overexpression contributes to the formation of immunosuppressive tumor microenvironment and is related to exacerbated prognosis, increased risk of metastasis and resistance to immunotherapy of various tumors. However, the prognostic significance of NT5E in pan-cancer is obscure so far. Methods: We explored the expression level of NT5E in cancers and adjacent tissues and revealed the relationship between the NT5E expression level and clinical outcomes in pan-cancer by utilizing the UCSC Xena database. Then, correlation analyses were performed to evaluate the relationship between NT5E expression and immune infiltration level via EPIC, MCP-counter and CIBERSORT methods, and the enrichment analysis were employed to identify NT5E-interacting molecules and functional pathways. Furthermore, we conducted single-cell analysis to explore the potential role of NT5E on single-cell level based on the CancerSEA database. Meanwhile, gene set enrichment analysis (GSEA) in single-cell level was also conducted in TISCH database and single-cell signature explorer was utilized to evaluate the epithelial-mesenchymal transition (EMT) level in each cell type. Results: The expression level of NT5E was aberrant in almost all cancer types, and was correlated with worse prognosis in several cancers. Notably, NT5E overexpression was related to worse overall survival (OS) in pancreatic adenocarcinoma (PAAD), head and neck squamous cell carcinoma (HNSC), mesothelioma (MESO), stomach adenocarcinoma (STAD), uveal melanoma (UVM) and cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) (p < 0.01). NT5E-related immune microenvironment analysis revealed that NT5E is associated positively with the degree of infiltration of cancer-associated fibroblasts (CAFs) and endothelial cells in most cancers. Enrichment analysis of cellular component (CC) demonstrated the critical part of NT5E played in cell-substrate junction, cell-substrate adherens junction, focal adhesion and external side of plasma membrane. Finally, single-cell analysis of NT5E illuminated that EMT function of CAFs was elevated in basal cell carcinoma (BCC), skin cutaneous melanoma (SKCM), HNSC and PAAD. Conclusion: NT5E could serve as a potential prognostic biomarker for cancers. The potential mechanism may be related to the upregulated EMT function of CAFs, which provides novel inspiration for immunotherapy by targeting CAFs with high NT5E expression.

Unraveling the Role and Spatial Architecture of Cancer-Associated Fibroblasts in Classical Hodgkin Lymphoma

Author response: Comprehensive characterization of tumor microenvironment in colorectal cancer via molecular analysis

Cancer stem cells (CSCs) play pivotal roles in tumor growth and therapeutic response; hence it is of fundamental importance to understand how CSCs is regulated inside the tumor mass. Similar to the regulation of normal stem cells by their ‘niche’, CSCs are also regulated by cells within the tumor microenvironment. Although most cases of HCC develop within a background of cirrhosis in which liver tissue is enriched in activated myofibroblasts, the role of cancer associated fibroblasts (CAFs) on liver CSCs within the tumor microenvironment has not yet studied. In order to achieve the goal of developing an effective therapy for HCC by specifically eliminating liver CSCs, we aim to dissect the cross-talks between HCC cells and CAFs. To study this, CAFs from fresh HCC tissues was successfully isolated, cultured and characterized by α-SMA immunoreactivity. Conditioned medium from CAFs enriched the populations of liver CSCs as reflected by an increased ability of self-renewal, chemoresistance, invasiveness and expression of stemness-associated genes and markers CD44 and CD47; and its CSC- stimulating effect was further enhanced when CAFs was stimulated with the conditioned medium of HCC cells. Using cytokine antibody array, hepatocyte growth factor (HGF) was found to be preferentially secreted by CAFs and increased upon stimulation by HCC cells. To verify whether the effect of CAFs on regulation of liver CSCs is due to HGF, the effect of recombinant HGF on regulation of liver CSCs was examined. Recombinant HGF at the physiological levels of CAFs (2ng/mL and 10ng/mL) promoted stem-like properties of HCC cells, and the effect of CAFs on CSC properties was alleviated with addition of HGF neutralizing antibody and c-met inhibitor (PHA665-752). In a cohort of HCC patient’s samples, HGF expression was significantly correlated with α-SMA (p=0.0003), suggesting the paracrine effect of CAFs on HCC cells. Further experiments showed that HCC cells induced proliferation and migration of CAFs to tumor-associated stroma; and CAFs in turn secretes HGF to regulate the CSC plasticity by activating NF-κB-mediated EMT transition. This study potentially dissects the signaling cross-talks between HCC cells and CAFs within the tumor microenvironment which may potentially open a new avenue to develop targeted therapy against this deadly disease. Citation Format: Yuen Ting Lau, Jessica Lo, Irene Oi Lin Ng, Terence Kin Wah Lee. Cancer associated fibroblasts-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2014-3044

Cancer-associated fibroblasts (CAFs) are integral components of the tumor microenvironment playing key roles in tumor progression, metastasis, and therapeutic resistance. However, challenges persist in understanding their heterogeneity, origin, and functional diversity. One major obstacle is the lack of standardized naming conventions for CAF subpopulations, with current systems failing to capture their full complexity. Additionally, the identification of CAFs is hindered by the absence of specific biomarkers, limiting the precision of diagnostic and therapeutic strategies. In vitro culture conditions often fail to maintain the in vivo characteristics of CAFs, which complicates their study and the translation of findings to clinical practice. Although current detection methods, such as antibodies, mRNA probes, and single-cell transcriptomics, offer insights into CAF biology, they lack standardization and fail to provide reliable quantitative measures. Furthermore, the dynamic interactions between CAFs, tumor cells, and immune cells within the TME remain insufficiently understood, and the role of CAFs in immune evasion and therapy resistance is an area of ongoing research. Understanding how CAFs influence drug resistance and the immune response is essential for developing more effective cancer therapies. This review aims to provide an in-depth analysis of the challenges in CAF research, propose future research directions, and emphasize the need for improved CAF-targeted therapeutic strategies. By addressing these gaps, it seeks to highlight the potential of CAFs as targets for overcoming therapeutic resistance and enhancing the efficacy of cancer treatments.

The dendritic cell (DC) -based vaccine immunotherapy has been a promising cancer immunotherapy, but has been insufficient to eradicate the tumor in patients with advanced cancer. This can result from the complicated tumor microenvironment (TME) that is implicated in suppression of anti-tumor immune responses. Several immune cell types in TME, such as Tregs, myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), have been reported to regulate anti-tumor immune responses negatively. Cancer-associated fibroblasts (CAFs) are also primary stromal cells in TME, and contribute to tumor growth and metastases through the secretion of TGF-β and stromal cell-derived factor-1 (SDF-1). We considered that TME-targeted strategies should be innovated for the development of the potent cancer immunotherapy. On the basis of these viewpoints, we focused on the role of CAFs in TME, and hypothesized that inhibition of CAFs would lead to improvement of systemic anti-tumor immune responses and enhancement of the potency of the DCs-based vaccine immunotherapy. In this study, we applied tranilast in order to inhibit CAFs, the anti-fibrotic and -allergic agent that is used clinically and has been shown to inhibit fibroblast in the scar tissue. In in vitro studies, we examined effects of tranilast on CAFs that were isolated from established EG7 (mouse lymphoma cells) tumors. As results, tranilast was able to suppress the proliferation of CAFs, and decrease the production of SDF-1 as well as TGF-β from CAFs. Regarding the effect on Tregs, tranilast was able to decrease the induction of them from spleen cells of normal mice. Based on these results, we confirmed that tranilast could inhibit the function of CAFs. Next, we examined the association between inhibition of CAFs and anti-tumor immune responses in tumor-bearing mouse model. C57BL/6 mice bearing EG7 were administered tranilast into the established tumor in combination with tumor antigen-loaded DCs vaccination, and were evaluated anti-tumor immune responses. As results, the population of CAFs was decreased by targeting them, leading to lower expressions of TGF-β as well as SDF-1 in TME. Inhibition of CAFs in TME resulted in the decreased distributions of Tregs in TME and tumor-draining lymph nodes (TDLs). On the induction of effector cells, antigen-specific CD8+ cells producing IFN-γ were significantly increased in TDLs and spleen through inhibition of CAFs in TME. In these mice, systemic antigen-specific cytotoxic responses were augmented, leading to suppression of tumor growth as compared with mice in control groups. These results demonstrate that CAFs are associated with immune suppression, and inhibition of CAFs functions in TME can augment systemic anti-tumor immune responses. Our mouse models provide a new rationale with TME-targeted strategies for enhancing the potency of the DCs-based vaccine immunotherapy. Citation Format: Yasuhiko Ohshio, Ryosuke Kaku, Keiko Ishida, Masayuki Hashimoto, Shoji Kitamura, Koji Teramoto, Jun Hanaoka, Noriaki Tezuka. The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2013-4945

CAFs (cancer-associated fibroblasts) are highly flexible cells of the cancer microenvironment. They produce the extracellular matrix (ECM) constituents that form the structure of the tumor stroma but are also a source of metabolites, growth factors, chemokines, and exosomes that impact every aspect of the tumor, including its response to treatment. It is believed that exosomal miRNAs facilitate intercellular signaling, which is essential for the development of cancer. The role of miRNAs and CAFs in the tumor microenvironment (TME) and carcinogenesis is reviewed in this paper. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) 2020 guidelines were used to perform a systematic review. Several databases, including Web of Science, Medline, Embase, Cochrane Library, and Scopus, were searched using the following keywords: CAFs, CAF, cancer-associated fibroblasts, stromal fibroblasts, miRNA, exosomal miRNAs, exosome and similar terms. We identified studies investigating exosomal miRNAs and CAFs in the TME and their role in carcinogenesis. A total of 12,572 papers were identified. After removing duplicates (n = 3803), 8774 articles were screened by title and abstract. Of these, 421 were excluded from further analysis. It has been reported that if exosomal miRNAs in CAFs are not functioning correctly, this may influence the secretory phenotype of tip cells and contribute to increased tumor invasiveness, tumor spread, decreased treatment efficacy, and a poorer prognosis. Under their influence, normal fibroblasts (NFs) are transformed into CAFs. Furthermore, they participate in metabolic reprogramming, which allows for fast proliferation of the cancer cell population, adaptation to growing energy demands, and the capacity to avoid immune system identification.

Background: A hallmark signature of the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) is abundantly infiltration of cancer-associated fibroblasts (CAFs), which facilitate HNSCC progression. However, some clinical trials showed targeted CAFs ended in failure, even accelerated cancer progression. Therefore, comprehensive exploration of CAFs should solve the shortcoming and facilitate the CAFs targeted therapies for HNSCC.Methods: In this study, we identified two CAFs gene expression patterns and performed the single‐sample gene set enrichment analysis (ssGSEA) to quantify the expression and construct score system. We used multi-methods to reveal the potential mechanisms of CAFs carcinogenesis progression. Finally, we integrated 10 machine learning algorithms and 107 algorithm combinations to construct most accurate and stable risk model. The machine learning algorithms contained random survival forest (RSF), elastic network (Enet), Lasso, Ridge, stepwise Cox, CoxBoost, partial least squares regression for Cox (plsRcox), supervised principal components (SuperPC), generalised boosted regression modelling (GBM), and survival support vector machine (survival-SVM).Results: There are two clusters present with distinct CAFs genes pattern. Compared to the low CafS group, the high CafS group was associated with significant immunosuppression, poor prognosis, and increased prospect of HPV negative. Patients with high CafS also underwent the abundant enrichment of carcinogenic signaling pathways such as angiogenesis, epithelial mesenchymal transition, and coagulation. The MDK and NAMPT ligand–receptor cellular crosstalk between the cancer associated fibroblasts and other cell clusters may mechanistically cause immune escape. Moreover, the random survival forest prognostic model that was developed from 107 machine learning algorithm combinations could most accurately classify HNSCC patients.Conclusion: We revealed that CAFs would cause the activation of some carcinogenesis pathways such as angiogenesis, epithelial mesenchymal transition, and coagulation and revealed unique possibilities to target glycolysis pathways to enhance CAFs targeted therapy. We developed an unprecedentedly stable and powerful risk score for assessing the prognosis. Our study contributes to the understanding of the CAFs microenvironment complexity in patients with head and neck squamous cell carcinoma and serves as a basis for future in-depth CAFs gene clinical exploration.

The tumor microenvironment (TME) is a significant contributor to cancer progression containing complex connections between cellular and chemical components and provides a suitable substrate for tumor growth and development. Growing evidence shows targeting tumor cells while ignoring the surrounding TME is not effective enough to overcome the cancer disease. Fibroblasts are essential sentinels of the stroma that due to certain conditions in TME, such as oxidative stress and local hypoxia, become activated, and play the prominent role in the physical support of tumor cells and the enhancement of tumorigenesis. Activated fibroblasts in TME, defined as cancer-associated fibroblasts (CAFs), play a crucial role in regulating the biological behavior of tumors, such as tumor metastasis and drug resistance. CAFs are highly heterogeneous populations that have different origins and, in addition to their role in supporting stromal cells, have multiple immunosuppressive functions via a membrane and secretory patterns. The secretion of different cytokines/chemokines, interactions that mediate the recruitment of regulatory immune cells and the reprogramming of an immunosuppressive function in immature myeloid cells are just a few examples of how CAFs contribute to the immune escape of tumors through various direct and indirect mechanisms on specific immune cell populations. Moreover, CAFs directly abolish the role of cytotoxic lymphocytes. The activation and overexpression of inhibitory immune checkpoints (iICPs) or their ligands in TME compartments are one of the main regulatory mechanisms that inactivate tumor-infiltrating lymphocytes in cancer lesions. CAFs are also essential players in the induction or expression of iICPs and the suppression of immune response in TME. Based on available studies, CAF subsets could modulate immune cell function in TME through iICPs in two ways; direct expression of iICPs by activated CAFs and indirect induction by production soluble and then upregulation of iICPs in TME. With a focus on CAFs’ direct and indirect roles in the induction of iICPs in TME as well as their use in immunotherapy and diagnostics, we present the evolving understanding of the immunosuppressive mechanism of CAFs in TME in this review. Understanding the complete picture of CAFs will help develop new strategies to improve precision cancer therapy.

Heterogeneity and function of cancer-associated fibroblasts in renal cell carcinoma

Tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) has a major influence on disease progression and therapy response. One of the predominant stromal cell types in the TME of HNSCC is cancer-associated fibroblasts (CAF). CAF constitute a diverse cell population and we are only at the beginning of characterizing and understanding the functions of various CAF subsets. CAF have been shown to interact with tumor cells and other components of the TME to shape mainly a favourable microenvironment for HNSCC progression, although some studies report existence of tumor-restraining CAF subtypes. The numerous pathways used by CAF to promote tumorigenesis may represent potential therapeutic targets. This review summarizes current knowledge on the origins, subtypes and mechanisms employed by CAF in HNSCC. The aim is to contribute to the understanding on how CAF actively influence the TME and modulate different immune cell types, as well as cancer cells, to establish a conducive setting for cancer growth. Although CAF are currently a promising therapeutic target for the treatment of other types of cancer, there is no significant therapeutic advancement in HNSCC.

There has been an increase in the amount of studies focusing on the relevance of tumour microenvironment (TME) in metastasis of breast cancer. Cancer-associated fibroblasts (CAFs) is one of the most abundant cells found in the TME that play key roles in the metastasis of cancers particularly in invasive ductal carcinoma (IDC), a common type of breast cancer. In a bid to constantly find novel treatments to address limited number of therapy that are currently available, CAFs have emerged as a target for many researches. This review aims to explore the multifaceted role of CAFs in IDC progression, highlighting their involvement in tumour growth, angiogenesis, epithelial-to-mesenchymal transition (EMT), immune modulation, and drug resistance. Additional focus was also placed on potential therapeutic benefit of targeting CAFs. A comprehensive review of recent studies on the subject CAF biology, CAF interactions within the TME, and existing therapeutic approaches targeting CAFs in IDC was conducted. Findings show that CAFs are crucial to the dynamics of the TME and they influence tumour progression. CAFs facilitate tumour progression through various mechanisms, some of which are: secretion of pro-tumorigenic factors and ECM remodelling. CAFs also play a part in suppression of immune responses. CAF-targeted drugs have shown promising results in treatment for patients. Mechanism of CAF-targeting drugs is through inhibition of TGF-β signalling and reprogramming of CAFs. However, challenges of CAF heterogeneity and off-target effects will have to be addressed. Interestingly, CAF being a key mediator of the TME in cancer also present potential diagnostic and prognostic value to assesses IDC progression. Targeting CAFs offers a promising approach for IDC diagnosis and treatment, although more research is needed to refine strategies for selective CAF targeting and overcoming therapeutic challenges.

Breast cancer is one of the leading causes of cancer related deaths in females. Current treatment options for breast cancer ultimately fail, resulting in cancer relapse. One explanation for recurrence after treatment is the presence of cancer stem cells (CSCs). CSCs have high proliferative potential and are able to initiate tumor growth. In order to target CSCs, it is important to understand how CSCs are maintained. Recent studies have shown that cancer‐associated fibroblasts (CAFs) aid in the maintenance of CSCs. CAFs are an abundant cell type in the tumor microenvironment and have been found to promote tumor growth and confer resistance to anti‐cancer drugs by supporting cancer stem cells. We hypothesized that CAFs promote and maintain the growth of cancer stem cells in a novel tumor model (MCaP). Mammary carcinoma cells (MCaP) were propagated in culture from a tumor that developed spontaneously in an aged mouse. Cancer cells, along with CAFs were expanded from the isolated tumor. Cancer cells were grown as a mixed culture with CAFs or alone after enrichment. A colony growth assay was used to assess the formation of CSCs, which grow as colonies. Cultures were fixed and stained with cytokeratin and colonies were counted manually. Colony size was quantified using ImageJ. Flow cytometry was performed to detect and quantify surface expression of CSC markers CD44 and CD24 in mixed cultures and isolated cancer cells. To investigate whether CAFs enable cancer cell resistance to chemotherapy, we measured the dose response to cyclophosphamide or paclitaxel. Immunofluorescence was performed on tissue sections of metastatic lymph node and primary tumor. Tissues were stained with cytokeratin, aldehyde dehydrogenase (ALDH), and alpha‐sma (SMA). From the colony growth assay we noticed that colony formation and the size of colonies were both increased when MCaP cells were cultured with CAFs. Flow cytometry showed that MCaP cancer cells grown with CAFs resulted in the appearance of a CD44 high/CD24low population. This CSC population was not seen in cancer cells grown alone. After treating cells with paclitaxel or cyclophosphamide, we saw no notable difference in the viability of cancer cells in the presence or absence of CAFs. Immunofluorescence staining showed ALDH was expressed in SMA‐positive CAFs, but not in cytokeratin‐positive cancer cells. Although we did not find that CAFs protected cancer cells from chemotherapy, CAFs appear to have a positive effect on the size and quantity of CSC colonies. In addition, we detected a CD44 high, CD24 low CSC population in mixed cultures. CAFs were found to express the CSC marker ALDH, with expression increasing as the size of the tumor increased. This finding suggests that CAFs may provide a niche for CSCs. In conclusion, these results suggest that CAFs play an important role in maintaining CSCs in the MCaP mouse model.Support or Funding InformationThe University of the Virgin Islands MARC grant (GM008422)and The Jones lab ACS Pilot grant (K22CA230315)