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Gareth J Thomas - One of the best experts on this subject based on the ideXlab platform.
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characterising Cancer Associated Fibroblast heterogeneity in non small cell lung Cancer a systematic review and meta analysis
Scientific Reports, 2021Co-Authors: Andrew F Irvine, Sara Waise, Edward W Green, Beth Stuart, Gareth J ThomasAbstract:Cancer-Associated Fibroblasts (CAFs) are a key component of the tumour microenvironment with evidence suggesting they represent a heterogeneous population. This study summarises the prognostic role of all proteins characterised in CAFs with immunohistochemistry in non-small cell lung Cancer thus far. The functions of these proteins in cellular processes crucial to CAFs are also analysed. Five databases were searched to extract survival outcomes from published studies and statistical techniques, including a novel method, used to capture missing values from the literature. A total of 26 proteins were identified, 21 of which were combined into 7 common cellular processes key to CAFs. Quality assessments for sensitivity analyses were carried out for each study using the REMARK criteria whilst publication bias was assessed using funnel plots. Random effects models consistently identified the expression of podoplanin (Overall Survival (OS)/Disease-specific Survival (DSS), univariate analysis HR 2.25, 95% CIs 1.80-2.82) and α-SMA (OS/DSS, univariate analysis HR 2.11, 95% CIs 1.18-3.77) in CAFs as highly prognostic regardless of outcome measure or analysis method. Moreover, proteins involved in maintaining and generating the CAF phenotype (α-SMA, TGF-β and p-Smad2) proved highly significant after sensitivity analysis (HR 2.74, 95% CIs 1.74-4.33) supporting attempts at targeting this pathway for therapeutic benefit.
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abstract 3762 single cell analysis of Cancer Associated Fibroblast heterogeneity in non small cell lung Cancer mapping molecular phenotypes in tumors
Cancer Research, 2019Co-Authors: Sara Waise, Christopher J Hanley, Rachel Parker, Christian H Ottensmeier, Matthew Rosezerilli, Gareth J ThomasAbstract:This work aims to characterise the heterogeneity and spatial relationships of the Cancer-Associated Fibroblast (CAF) population in non-small cell lung Cancer. Fresh human lung tissue was dissociated for sixty minutes to extract the maximum possible proportion of Fibroblasts. Single-cell RNA sequencing was performed using a droplet-barcoded platform (Drop-seq). Quality control was performed on the raw sequencing data and resulting gene expression matrix. Bioinformatic analysis was performed using multiple packages in R. Spatial relationships between cell types were assessed using a multi-immunohistochemical (IHC) staining technique. We developed a workflow for efficient processing of raw Drop-seq data including quality control, normalisation and visualisation. Low-quality events were identified by integrating previously-described and novel quality-control metrics into a machine learning (random forest) model, and demonstrated that this approach improves clustering quality. Applying this method to samples from twelve non-small cell lung Cancer (NSCLC) patients, we identified 5 distinct Fibroblast subtypes; 3 predominantly derived from normal tissue and 2 largely from tumor samples. Of the normal subtypes, one showed gene expression consistent with the previously-described "inflammatory" Fibroblast phenotype. Trajectory analysis identified a branched differentiation process from normal to CAF phenotypes, suggesting that these cells share a common initial activation before differentiation to either a "matrix remodelling" or "hypoxic" subtype. The prevalence and impact of these sub-populations appears to differ between NSCLC subtypes. The "matrix remodelling" subtype is present in both adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), but confers a negative prognostic effect in LUAD only; the "hypoxic" phenotype appears relatively LUSC-specific. Multiplexed IHC using identified cluster markers demonstrated that these subtypes have different spatial distributions and relationships to other cell types. CAF remain a poorly-characterised population, despite their abundance in most solid Cancers. No single molecular marker identifies all CAF, and there has been a scarcity of evidence regarding the existence of distinct subtypes and whether such subgroups have different functions. Our analysis has revealed five distinct CAF subtypes in NSCLC. In addition to divergent differentiation pathways, these subtypes have differential gene set enrichment, indicative of functional differences. In keeping with this, the phenotypes show distinct prognostic impact across NSCLC subtypes. Characterisation of CAF subgroups Associated with aggressive tumor progression may facilitate identification of novel stromal targeting strategies. Citation Format: Sara Waise, Christopher J. Hanley, Rachel Parker, Christian H. Ottensmeier, Matthew Rose-Zerilli, Gareth J. Thomas. Single-cell analysis of Cancer-Associated Fibroblast heterogeneity in non-small cell lung Cancer: Mapping molecular phenotypes in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3762.
Sara Waise - One of the best experts on this subject based on the ideXlab platform.
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characterising Cancer Associated Fibroblast heterogeneity in non small cell lung Cancer a systematic review and meta analysis
Scientific Reports, 2021Co-Authors: Andrew F Irvine, Sara Waise, Edward W Green, Beth Stuart, Gareth J ThomasAbstract:Cancer-Associated Fibroblasts (CAFs) are a key component of the tumour microenvironment with evidence suggesting they represent a heterogeneous population. This study summarises the prognostic role of all proteins characterised in CAFs with immunohistochemistry in non-small cell lung Cancer thus far. The functions of these proteins in cellular processes crucial to CAFs are also analysed. Five databases were searched to extract survival outcomes from published studies and statistical techniques, including a novel method, used to capture missing values from the literature. A total of 26 proteins were identified, 21 of which were combined into 7 common cellular processes key to CAFs. Quality assessments for sensitivity analyses were carried out for each study using the REMARK criteria whilst publication bias was assessed using funnel plots. Random effects models consistently identified the expression of podoplanin (Overall Survival (OS)/Disease-specific Survival (DSS), univariate analysis HR 2.25, 95% CIs 1.80-2.82) and α-SMA (OS/DSS, univariate analysis HR 2.11, 95% CIs 1.18-3.77) in CAFs as highly prognostic regardless of outcome measure or analysis method. Moreover, proteins involved in maintaining and generating the CAF phenotype (α-SMA, TGF-β and p-Smad2) proved highly significant after sensitivity analysis (HR 2.74, 95% CIs 1.74-4.33) supporting attempts at targeting this pathway for therapeutic benefit.
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abstract 3762 single cell analysis of Cancer Associated Fibroblast heterogeneity in non small cell lung Cancer mapping molecular phenotypes in tumors
Cancer Research, 2019Co-Authors: Sara Waise, Christopher J Hanley, Rachel Parker, Christian H Ottensmeier, Matthew Rosezerilli, Gareth J ThomasAbstract:This work aims to characterise the heterogeneity and spatial relationships of the Cancer-Associated Fibroblast (CAF) population in non-small cell lung Cancer. Fresh human lung tissue was dissociated for sixty minutes to extract the maximum possible proportion of Fibroblasts. Single-cell RNA sequencing was performed using a droplet-barcoded platform (Drop-seq). Quality control was performed on the raw sequencing data and resulting gene expression matrix. Bioinformatic analysis was performed using multiple packages in R. Spatial relationships between cell types were assessed using a multi-immunohistochemical (IHC) staining technique. We developed a workflow for efficient processing of raw Drop-seq data including quality control, normalisation and visualisation. Low-quality events were identified by integrating previously-described and novel quality-control metrics into a machine learning (random forest) model, and demonstrated that this approach improves clustering quality. Applying this method to samples from twelve non-small cell lung Cancer (NSCLC) patients, we identified 5 distinct Fibroblast subtypes; 3 predominantly derived from normal tissue and 2 largely from tumor samples. Of the normal subtypes, one showed gene expression consistent with the previously-described "inflammatory" Fibroblast phenotype. Trajectory analysis identified a branched differentiation process from normal to CAF phenotypes, suggesting that these cells share a common initial activation before differentiation to either a "matrix remodelling" or "hypoxic" subtype. The prevalence and impact of these sub-populations appears to differ between NSCLC subtypes. The "matrix remodelling" subtype is present in both adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), but confers a negative prognostic effect in LUAD only; the "hypoxic" phenotype appears relatively LUSC-specific. Multiplexed IHC using identified cluster markers demonstrated that these subtypes have different spatial distributions and relationships to other cell types. CAF remain a poorly-characterised population, despite their abundance in most solid Cancers. No single molecular marker identifies all CAF, and there has been a scarcity of evidence regarding the existence of distinct subtypes and whether such subgroups have different functions. Our analysis has revealed five distinct CAF subtypes in NSCLC. In addition to divergent differentiation pathways, these subtypes have differential gene set enrichment, indicative of functional differences. In keeping with this, the phenotypes show distinct prognostic impact across NSCLC subtypes. Characterisation of CAF subgroups Associated with aggressive tumor progression may facilitate identification of novel stromal targeting strategies. Citation Format: Sara Waise, Christopher J. Hanley, Rachel Parker, Christian H. Ottensmeier, Matthew Rose-Zerilli, Gareth J. Thomas. Single-cell analysis of Cancer-Associated Fibroblast heterogeneity in non-small cell lung Cancer: Mapping molecular phenotypes in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3762.
Paolo G Dotto - One of the best experts on this subject based on the ideXlab platform.
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notch1 gene amplification promotes expansion of Cancer Associated Fibroblast populations in human skin
Nature Communications, 2020Co-Authors: Atul Katarkar, Pino Bordignon, Paola Ostano, Sandro Goruppi, Andrea Clocchiatti, Giulia Bottoni, Francesca Lazzaroni, Ilaria Gregnanin, Victor A Neel, Paolo G DottoAbstract:Cancer Associated Fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal Fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses Cancer/stromal cells expansion. Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-Cancer intervention. The presence of genomic alterations in Cancer Associated Fibroblasts (CAFs) is largely unexplored. The authors show that frequent NOTCH1 gene amplification and overexpression render CAFs resistant to the UVA-induced DNA damage response (DDR) and promote Cancer/stromal cells expansion, which can be reversed by NOTCH inhibition.
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the ulk3 kinase is critical for convergent control of Cancer Associated Fibroblast activation by csl and gli
Cell Reports, 2017Co-Authors: Sandro Goruppi, Maria Giuseppina Procopio, Andrea Clocchiatti, Paolo G Dotto, Victor A NeelAbstract:The connection between signaling pathways activating Cancer-Associated Fibroblasts (CAFs) remains to be determined. Metabolic alterations linked to autophagy have also been implicated in CAF activation. CSL/RBPJ, a transcriptional repressor that mediates Notch signaling, suppresses the gene expression program(s), leading to stromal senescence and CAF activation. Deregulated GLI signaling can also contribute to CAF conversion. Here, we report that compromised CSL function depends on GLI activation for conversion of human dermal Fibroblasts into CAFs, separately from cellular senescence. Decreased CSL upregulates the expression of the ULK3 kinase, which binds and activates GLI2. Increased ULK3 also induces autophagy, which is unlinked from GLI and CAF activation. ULK3 upregulation occurs in the CAFs of several tumor types, and ULK3 silencing suppresses the tumor-enhancing properties of these cells. Thus, ULK3 links two key signaling pathways involved in CAF conversion and is an attractive target for stroma-focused anti-Cancer intervention.
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convergent roles of atf3 and csl in chromatin control of Cancer Associated Fibroblast activation
Journal of Experimental Medicine, 2017Co-Authors: Dong-eun Kim, Maria Giuseppina Procopio, Pino Bordignon, Sandro Goruppi, Paolo G Dotto, Victor A Neel, Soumitra Ghosh, Francesco Magliozzi, P AngelinoAbstract:Cancer-Associated Fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a stress-responsive transcriptional repressor, is down-modulated in skin Cancer stromal cells, and Atf3 knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of IL6 induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous Cancer-stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to Cancer- and stroma-focused intervention.
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pdcd4 is a csl Associated protein with a transcription repressive function in Cancer Associated Fibroblast activation
Oncotarget, 2016Co-Authors: Dong-eun Kim, Andrea Clocchiatti, Paolo G DottoAbstract:// Seung-Hee Jo 1,2 , Dong Eun Kim 3 , Andrea Clocchiatti 1,2 and G. Paolo Dotto 1,3 1 Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA, USA 2 Department of Dermatology, Harvard Medical School, Boston, MA, USA 3 Department of Biochemistry, University of Lausanne, Epalinges, CH, Switzerland Correspondence to: G. Paolo Dotto, email: // Keywords : PDCD4, Notch/CSL signaling, transcription repression, squamous Cancer, CAFs Received : May 12, 2016 Accepted : July 22, 2016 Published : August 11, 2016 Abstract The Notch/CSL pathway plays an important role in skin homeostasis and carcinogenesis. CSL, the key effector of canonical Notch signaling endowed with an intrinsic transcription repressive function, suppresses stromal Fibroblast senescence and Cancer Associated Fibroblast (CAF) activation through direct down-modulation of key effector genes. Interacting proteins that participate with CSL in this context are as yet to be identified. We report here that Programmed Cell Death 4 (PDCD4), a nuclear/cytoplasmic shuttling protein with multiple functions, associates with CSL and plays a similar role in suppressing dermal Fibroblast senescence and CAF activation. Like CSL, PDCD4 is down-regulated in stromal Fibroblasts of premalignant skin actinic keratosis (AKs) lesions and squamous cell carcinoma (SCC). While devoid of intrinsic DNA binding capability, PDCD4 is present at CSL binding sites of CAF marker genes as well as canonical Notch/CSL targets and suppresses expression of these genes in a Fibroblast-specific manner. Thus, we propose that PDCD4 is part of the CSL repressive complex involved in negative control of stromal Fibroblasts conversion into CAFs.
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abstract ia10 convergent control of Cancer Associated Fibroblast activation and field Cancerization by notch csl and p53 signaling
Cancer Research, 2016Co-Authors: Paolo G DottoAbstract:The vast majority of epithelial Cancers is limited to in situ lesions that, for internal organs like breast, prostate or lung, can remain undetected for the whole life of an individual. The reason(s) why only a minor fraction of these lesions progresses into malignancy is not understood. Changes in tumor stroma are most frequently viewed as secondary to changes in the epithelium. However, recent evidence indicates that they may play a primary role. Such a possibility would help explain not only dormancy of most epithelial Cancers, but also field Cancerization, a condition of major clinical significance linked with multifocal and recurrent tumors and broader tissue changes beyond areas of tumor development that expand over time. Stromal Fibroblast senescence has been linked to aging-Associated Cancer risk. However, density and proliferation of Cancer Associated Fibroblasts (CAF) are frequently increased. In the mouse, deletion of the Notch effector CSL/RBP-Jκ in dermal Fibroblasts is sufficient for CAF activation and ensuing multifocal keratinocyte tumors. We report that CSL silencing induces senescence of primary Fibroblasts of both mouse and human origin, and from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal Fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes Fibroblast senescence, enhances expression of CAF effectors and promotes stromal and Cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. Citation Format: G. Paolo Dotto, G. Paolo Dotto. Convergent control of Cancer Associated Fibroblast activation and field Cancerization by Notch/CSL and p53 signaling. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA10.
Ja Seung Koo - One of the best experts on this subject based on the ideXlab platform.
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expression of Cancer Associated Fibroblast related proteins differs between invasive lobular carcinoma and invasive ductal carcinoma
Breast Cancer Research and Treatment, 2016Co-Authors: Cheol Keun Park, Woo Hee Jung, Ja Seung KooAbstract:Cancer-Associated Fibroblasts (CAFs) are classified into various functional subtypes such as Fibroblast activation protein-α (FAP-α), Fibroblast specific protein-1 (FSP-1), platelet-derived growth factor receptor-α (PDGFR-α), and PDGFR-β. In this study, we compared the expression of CAF-related proteins in invasive lobular carcinoma (ILC) with those in invasive carcinoma of no special type (NST) and assessed the implications of the differences observed. Using tissue microarrays of 104 ILC and 524 invasive carcinoma (NST) cases, immunohistochemistry for CAF-related proteins [podoplanin, prolyl 4-hydroxylase, FAP-α, FSP-1/S100A4, PDGFR-α, PDGFR-β, and chondroitin sulfate proteoglycan (NG2)] was conducted. In invasive carcinoma (NST), tumor cells expressed a high level of PDGFR-α, whereas ILC tumor cells expressed high levels of podoplanin, prolyl 4-hydroxylase, FAP-α, and FSP-1/S100A4. In stromal cells of invasive carcinoma (NST), high expression levels of prolyl 4-hydroxylase, PDGFR-α, and NG2 were observed, whereas ILC stromal cells expressed high levels of FAP-α, FSP-1/S100A4, and PDGFR-β. In ILC, tumoral FSP-1/S100A4 positivity was Associated with higher Ki-67 labeling index (p = 0.010) and non-luminal A type Cancer (p = 0.014). Stromal PDGFR-α positivity was Associated with lymph node metastasis (p = 0.011). On survival analysis of entire cases, tumoral FSP-1/S100A4 positivity (p = 0.002), stromal podoplanin positivity (p = 0.041), and stromal FSP-1/S100A4 negativity (p = 0.041) were Associated with shorter disease-free survival; only tumoral FSP-1/S100A4 positivity (p = 0.044) was Associated with shorter overall survival. In ILC, the expression of FAP-α and FSP-1/S100A4 was higher in both tumor and stromal cells than that observed in invasive carcinoma (NST). These results indicate that CAFs are a potential target in ILC treatment.
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Expression of Cancer-Associated Fibroblast related proteins in metastatic breast Cancer: an immunohistochemical analysis
Journal of translational medicine, 2015Co-Authors: Hye Min Kim, Woo Hee Jung, Ja Seung KooAbstract:Background Cancer-Associated Fibroblast (CAF) is the most studied element of the tumor microenvironment, although no relationship has been identified between expression of their related proteins and the metastasis site. The purpose of this study was to investigate the expression of CAF related proteins and their implications according to the metastasis site in metastatic breast Cancer.
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expression of Cancer Associated Fibroblast related proteins in adipose stroma of breast Cancer
Tumor Biology, 2015Co-Authors: Yoon Yang Jung, Yu Kyung Lee, Ja Seung KooAbstract:Cancer-Associated Fibroblasts (CAFs) play key roles in tumor microenvironment; they are thought to originate from adipocytes. This study aimed to evaluate CAF-related protein expression and its implications in breast Cancer. Of the 939 enrolled breast Cancer patients, 642 had fibrous and 297 had adipose stroma. The status of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER-2), Ki-67, podoplanin, prolyl 4-hydroxylase, Fibroblast activation protein α (FAPα), S100A4, platelet-derived growth factor receptor α (PDGFRα), PDGFRβ, and chondroitin sulfate proteoglycan (NG2) was evaluated via tissue microarrays. Tumors were divided into luminal A, luminal B, HER-2, or triple-negative breast Cancer subtypes according to their molecular status. Luminal A subtype was more prevalent in breast Cancer of adipose stroma type, whereas other molecular subtypes were more common in fibrous stroma type (p < 0.001). Tumor cell expression of podoplanin and FAPα was higher in adipose stroma type, while higher expression of prolyl 4-hydroxylase and PDGFRα was observed in fibrous stroma type. Furthermore, adipose stroma type exhibited higher stromal expression of podoplanin, FAPα, PDGFRβ, and NG2, whereas fibrous stroma type had higher prolyl 4-hydroxylase and S100A4 expression. In adipose stroma type, tumor positivity (p = 0.034) and stromal positivity (p = 0.005) of prolyl 4-hydroxylase were Associated with shorter disease-free survival, and stromal prolyl 4-hydroxylase positivity was with shorter overall survival (p < 0.001). In conclusion, expression of CAF-related proteins was observed in breast Cancer, with different profiles between adipose and fibrous stroma types. Prolyl 4-hydrolase status might be of prognostic value in adipose stroma type.
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differential expression of Cancer Associated Fibroblast related proteins according to molecular subtype and stromal histology in breast Cancer
Breast Cancer Research and Treatment, 2015Co-Authors: Sung Yeon Park, Hye Min Kim, Ja Seung KooAbstract:The purpose of this study aimed to investigate the clinicopathologic characteristics of breast Cancer according to its Cancer-Associated Fibroblast (CAF) phenotype. Immunohistochemistry staining of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER-2), Ki-67, podoplanin, prolyl 4-hydroxylase, Fibroblast activation protein alpha (FAPα), S100A4, platelet-derived growth factor receptor alpha (PDGFRα), PDGFRβ, and chondroitin sulfate proteoglycan (NG2) was performed on tissue microarray consisting of 642 breast Cancer cases. Samples were categorized into luminal A, luminal B, HER-2, or triple-negative breast Cancer (TNBC) according to immunohistochemical results, whereas tumor stroma was classified into desmoplastic, sclerotic, normal-like, or inflammatory type based on histological findings. Expression of CAF-related proteins in the stroma differed depending on breast Cancer molecular subtypes. All CAF-related protein expression was high (p < 0.05) in HER-2 type, whereas in luminal A, the expression of FAPα, PDGFα, PDGFβ, and NG2 was low, and in TNBC, the expression of podoplanin, prolyl 4-hydroxylase, and S100A4 was low. In the stromal component, CAF-related protein expression differed according to stromal phenotype (p < 0.001). The desmoplastic type showed high expression of podoplanin, prolyl 4-hydroxylase, S100A4, PDGFRα, and PDGFRβ, whereas the sclerotic type exhibited low expression of FAPα, PDGFα, PDGFβ, and NG2. The inflammatory type had high expression of FAPα and NG2 with low podoplanin, while normal-like type showed low expression of prolyl 4-hydroxylase and S100A4. Our results suggested that differential CAF-related protein expression depended on the molecular subtypes and stromal histologic features of breast Cancer, indicating that in the future, this system could potentially use these markers for prognosis prediction and targeted therapy of breast Cancer.
Christopher J Hanley - One of the best experts on this subject based on the ideXlab platform.
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nox4 inhibition potentiates immunotherapy by overcoming Cancer Associated Fibroblast mediated cd8 t cell exclusion from tumors
Cancer Research, 2020Co-Authors: Kirsty Ford, Christopher J Hanley, Massimiliano Mellone, Cedric Szyndralewiez, Freddy Heitz, Philippe Wiesel, Oliver Wood, Maria Machado, Mariaantoinette Lopez, Anushapreethi GanesanAbstract:Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-Associated Fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [antiCancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human Cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of Cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion. GRAPHICAL ABSTRACT: http://Cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.See related commentary by Hayward, p. 1799.
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abstract 3762 single cell analysis of Cancer Associated Fibroblast heterogeneity in non small cell lung Cancer mapping molecular phenotypes in tumors
Cancer Research, 2019Co-Authors: Sara Waise, Christopher J Hanley, Rachel Parker, Christian H Ottensmeier, Matthew Rosezerilli, Gareth J ThomasAbstract:This work aims to characterise the heterogeneity and spatial relationships of the Cancer-Associated Fibroblast (CAF) population in non-small cell lung Cancer. Fresh human lung tissue was dissociated for sixty minutes to extract the maximum possible proportion of Fibroblasts. Single-cell RNA sequencing was performed using a droplet-barcoded platform (Drop-seq). Quality control was performed on the raw sequencing data and resulting gene expression matrix. Bioinformatic analysis was performed using multiple packages in R. Spatial relationships between cell types were assessed using a multi-immunohistochemical (IHC) staining technique. We developed a workflow for efficient processing of raw Drop-seq data including quality control, normalisation and visualisation. Low-quality events were identified by integrating previously-described and novel quality-control metrics into a machine learning (random forest) model, and demonstrated that this approach improves clustering quality. Applying this method to samples from twelve non-small cell lung Cancer (NSCLC) patients, we identified 5 distinct Fibroblast subtypes; 3 predominantly derived from normal tissue and 2 largely from tumor samples. Of the normal subtypes, one showed gene expression consistent with the previously-described "inflammatory" Fibroblast phenotype. Trajectory analysis identified a branched differentiation process from normal to CAF phenotypes, suggesting that these cells share a common initial activation before differentiation to either a "matrix remodelling" or "hypoxic" subtype. The prevalence and impact of these sub-populations appears to differ between NSCLC subtypes. The "matrix remodelling" subtype is present in both adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), but confers a negative prognostic effect in LUAD only; the "hypoxic" phenotype appears relatively LUSC-specific. Multiplexed IHC using identified cluster markers demonstrated that these subtypes have different spatial distributions and relationships to other cell types. CAF remain a poorly-characterised population, despite their abundance in most solid Cancers. No single molecular marker identifies all CAF, and there has been a scarcity of evidence regarding the existence of distinct subtypes and whether such subgroups have different functions. Our analysis has revealed five distinct CAF subtypes in NSCLC. In addition to divergent differentiation pathways, these subtypes have differential gene set enrichment, indicative of functional differences. In keeping with this, the phenotypes show distinct prognostic impact across NSCLC subtypes. Characterisation of CAF subgroups Associated with aggressive tumor progression may facilitate identification of novel stromal targeting strategies. Citation Format: Sara Waise, Christopher J. Hanley, Rachel Parker, Christian H. Ottensmeier, Matthew Rose-Zerilli, Gareth J. Thomas. Single-cell analysis of Cancer-Associated Fibroblast heterogeneity in non-small cell lung Cancer: Mapping molecular phenotypes in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3762.
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targeting the myoFibroblastic Cancer Associated Fibroblast phenotype through inhibition of nox4
Journal of the National Cancer Institute, 2018Co-Authors: Christopher J Hanley, Massimiliano Mellone, Kirsty Ford, Steve Thirdborough, Toby Mellows, Steven J Frampton, David M Smith, Elena Harden, Cedric Szyndralewiez, Marc D BullockAbstract:Background Cancer-Associated Fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many Cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods CAF accumulation and prognostic significance in head and neck Cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal Cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating Fibroblast-to-myoFibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results Patients with moderate/high levels of myoFibroblastic-CAF had a statistically significant decrease in Cancer-specific survival rates in each Cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myoFibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human Cancers (P < .001), strongly correlating with myoFibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myoFibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myoFibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across Cancer types.