FN1 and cancer-associated fibroblasts markers influence immune microenvironment in clear cell renal cell carcinoma.

BACKGROUND: Altered tumor microenvironment (TME) is characterized in clear cell renal cell carcinoma (ccRCC) as a result of the heterogeneity observed in the TME. Modulations in TME have shown tumor metastasis promotion; hence, identifying TME-based biomarkers can be critical for theranostics application. METHODS: Here, we performed an integrated systems biology approach utilizing differential gene expression, network metrics and clinical samples cohorts to prioritize the major deregulated genes and their associated pathways specific for metastasis. RESULTS: The gene expression profiling of 140 ccRCC samples resulted in 3657 differentially expressed genes, from which a network of 1867 up-regulated genes were further computed using network metrics for screening hub-genes. The specific pathways of ccRCC entailed through functional enrichment analysis of the hub-gene clusters indicated the role of the identified hub-genes in the enriched pathways, further validating the functional significance of the hub-genes. The positive correlation of TME cells, namely cancer-associated fibroblasts (CAFs) and its biomarkers (FAP and S100A4) with FN1, signified the role of hub-gene signaling for promoting metastasis in ccRCC. Thereafter, comparative expression, differential methylation, genetic alteration and overall survival analysis were analyzed to validate the screened hub-genes. CONCLUSIONS: The hub-genes were validated and prioritized by correlating with expression-based parameters, including histological grades, tumor, metastatic and pathological stages (based on median transcript per million; analysis of variance [ANOVA], P ≤ 0.05) from a clinically curated ccRCC dataset to further substantiate the translational benefits of the screened hub-genes as potential diagnostic biomarkers for ccRCC.

Alpha-smooth muscle actin-positive cancer-associated fibroblasts secreting osteopontin promote growth of luminal breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been shown to support tumor development in a variety of cancers. Different markers were applied to classify CAFs in order to elucidate their impact on tumor progression. However, the exact mechanism by which CAFs enhance cancer development and metastasis is yet unknown. METHODS: Alpha-smooth muscle actin (α-SMA) was examined immunohistochemically in intratumoral CAFs of nonmetastatic breast cancers and correlated with clinicopathological data. Four CAF cell lines were isolated from patients with luminal breast cancer (lumBC) and classified according to the presence of α-SMA protein. Conditioned medium (CM) from CAF cultures was used to assess the influence of CAFs on lumBC cell lines: MCF7 and T47D cells using Matrigel 3D culture assay. To identify potential factors accounting for promotion of tumor growth by α-SMAhigh CAFs, nCounter PanCancer Immune Profiling Panel (NanoString) was used. RESULTS: In luminal breast cancer, presence of intratumoral CAFs expressing high level of α-SMA (13% of lumBC group) correlated with poor prognosis (p = 0.019). In in vitro conditions, conditioned medium obtained from primary cultures of α-SMA-positive CAFs isolated from luminal tumors was observed to enhance growth of lumBC cell line colonies in 3D Matrigel, in contrast to CM derived from α-SMA-negative CAFs. Multigene expression analysis indicated that osteopontin (OPN) was overexpressed in α-SMA-positive CAFs in both clinical samples and in vitro models. OPN expression was associated with higher percentage of Ki67-positive cells in clinical material (p = 0.012), while OPN blocking in α-SMA-positive CAF-derived CM attenuated growth of lumBC cell line colonies in 3D Matrigel. CONCLUSIONS: Our findings demonstrate that α-SMA-positive CAFs might enhance tumor growth via secretion of OPN.

Dipeptidyl Peptidase-4 from Cancer-associated Fibroblasts Stimulates the Proliferation of Scirrhous-type Gastric Cancer Cells.

BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. MATERIALS AND METHODS: OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. RESULTS: The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. CONCLUSION: DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.

Three subtypes of lung cancer fibroblasts define distinct therapeutic paradigms.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Proteomics reveals the function reverse of MPSSS-treated prostate cancer-associated fibroblasts to suppress PC-3 cell viability via the FoxO pathway.

Prostate cancer-associated fibroblasts (prostate CAFs) are essential components of the tumor microenvironment and can promote tumor progression through their immunosuppressive functions. MPSSS, a novel polysaccharide purified from Lentinus edodes, has been reported to have anti-tumor activity. MPSSS could also inhibit the immunosuppressive function of prostate CAFs, which has been demonstrated through that the secretome of MPSSS-treated prostate CAFs could inhibit the proliferation of T cells. However, how the secretome of MPSSS-treated prostate CAFs influence prostate cancer progression is still unclear. Interestingly, we found that the low molecular weight (3-100kD) secretome of prostate CAFs (lmwCAFS) could promote the growth of PC-3 cells, while that of MPSSS-treated prostate CAFs (MT-lmwCAFS) could inhibit their growth. We carried out comparative secretomic analysis of lmwCAFS and MT-lmwCAFS to identify functional molecules that inhibit the growth of PC-3 cells, and proteomic analysis of lmwCAFS-treated PC-3 cells and MT-lmwCAFS-treated PC-3 cells to investigate the underlying molecular mechanism. These analyses suggest that TGF-ß3 from MT-lmwCAFS may inhibit the growth of PC-3 cells. The validated experiments revealed that TGF-ß3 from MT-lmwCAFS activated p21 expression in PC-3 cells by regulating the FoxO pathway thereby inducing G0/G1 cell cycle arrest of PC-3 cells. Overall, our data demonstrated that MPSSS reversed the ability of prostate CAFs to suppress the cell viability of PC-3 cells, which might provide a potential therapeutic strategy to prevent prostate cancer progression.

A distinct repertoire of cancer-associated fibroblasts is enriched in cribriform prostate cancer.

Outcomes for men with localized prostate cancer vary widely, with some men effectively managed without treatment on active surveillance, while other men rapidly progress to metastatic disease despite curative-intent therapies. One of the strongest prognostic indicators of outcome is grade groups based on the Gleason grading system. Gleason grade 4 prostate cancer with cribriform morphology is associated with adverse outcomes and can be utilized clinically to improve risk stratification. The underpinnings of disease aggressiveness associated with cribriform architecture are not fully understood. Most studies have focused on genetic and molecular alterations in cribriform tumor cells; however, less is known about the tumor microenvironment in cribriform prostate cancer. Cancer-associated fibroblasts (CAFs) are a heterogeneous population of fibroblasts in the tumor microenvironment that impact cancer aggressiveness. The overall goal of this study was to determine if cribriform prostate cancers are associated with a unique repertoire of CAFs. Radical prostatectomy whole-tissue sections were analyzed for the expression of fibroblast markers (ASPN in combination with FAP, THY1, ENG, NT5E, TNC, and PDGFRß) in stroma adjacent to benign glands and in Gleason grade 3, Gleason grade 4 cribriform, and Gleason grade 4 noncribriform prostate cancer by RNAscope®. Halo® Software was used to quantify percent positive stromal cells and expression per positive cell. The fibroblast subtypes enriched in prostate cancer were highly heterogeneous. Both overlapping and distinct populations of low abundant fibroblast subtypes in benign prostate stroma were enriched in Gleason grade 4 prostate cancer with cribriform morphology compared to Gleason grade 4 prostate cancer with noncribriform morphology and Gleason grade 3 prostate cancer. In addition, gene expression was distinctly altered in CAF subtypes adjacent to cribriform prostate cancer. Overall, these studies suggest that cribriform prostate cancer has a unique tumor microenvironment that may distinguish it from other Gleason grade 4 morphologies and lower Gleason grades.

Periostin as a key molecule defining desmoplastic environment in colorectal cancer.

Categorizing desmoplastic reaction (DR) based on the histological findings of cancer-associated fibroblasts is shown to be a promising novel method to predict prognosis of patients with colorectal cancer (CRC). Periostin (POSTN) in cancer-associated stroma is reportedly associated with poor clinical outcomes. Immunohistochemical staining with an anti-POSTN antibody was performed in 73 patients with pStage III CRC (cohort 1). In addition, to evaluate mRNA and protein expression levels of POSTN, we analyzed paired normal and invasive cancer frozen specimens by quantitative real-time polymerase chain reaction and western blot analysis in 41 patients (cohort 2). In cohort 1, according to the DR categorization, 18, 22, and 33 patients were classified as immature, intermediate, and mature, respectively. High immunoreactivity of POSTN was observed 100%, 68.2%, and 27.3%, respectively (p < 0.0001). The 5-year relapse-free survival rates were 56.8% and 82.7% in high and low POSTN expression subgroups, respectively (p = 0.015). In cohort 2, the POSTN mRNA and protein levels were significantly higher in the immature stroma as compared to the stroma characterized as other DR patterns. POSTN expression was closely associated with DR categorization. POSTN may be a key molecule that contributes to the malignant potential of CRC.

Alterations in the methylome of the stromal tumour microenvironment signal the presence and severity of prostate cancer.

BACKGROUND: Prostate cancer changes the phenotype of cells within the stromal microenvironment, including fibroblasts, which in turn promote tumour progression. Functional changes in prostate cancer-associated fibroblasts (CAFs) coincide with alterations in DNA methylation levels at loci-specific regulatory regions. Yet, it is not clear how these methylation changes compare across CAFs from different patients. Therefore, we examined the consistency and prognostic significance of genome-wide DNA methylation profiles between CAFs from patients with different grades of primary prostate cancer. RESULTS: We used Infinium MethylationEPIC BeadChips to evaluate genome-wide DNA methylation profiles from 18 matched CAFs and non-malignant prostate tissue fibroblasts (NPFs) from men with moderate to high grade prostate cancer, as well as five unmatched benign prostate tissue fibroblasts (BPFs) from men with benign prostatic hyperplasia. We identified two sets of differentially methylated regions (DMRs) in patient CAFs. One set of DMRs reproducibly differed between CAFs and fibroblasts from non-malignant tissue (NPFs and BPFs). Indeed, more than 1200 DMRs consistently changed in CAFs from every patient, regardless of tumour grade. The second set of DMRs varied between CAFs according to the severity of the tumour. Notably, hypomethylation of the EDARADD promoter occurred specifically in CAFs from high-grade tumours and correlated with increased transcript abundance and increased EDARADD staining in patient tissue. Across multiple cohorts, tumours with low EDARADD DNA methylation and high EDARADD mRNA expression were consistently associated with adverse clinical features and shorter recurrence free survival. CONCLUSIONS: We identified a large set of DMRs that are commonly shared across CAFs regardless of tumour grade and outcome, demonstrating highly consistent epigenome changes in the prostate tumour microenvironment. Additionally, we found that CAFs from aggressive prostate cancers have discrete methylation differences compared to CAFs from moderate risk prostate cancer. Together, our data demonstrates that the methylome of the tumour microenvironment reflects both the presence and the severity of the prostate cancer and, therefore, may provide diagnostic and prognostic potential.

Deficiency of the adrenomedullin-RAMP3 system suppresses metastasis through the modification of cancer-associated fibroblasts.

Tumor metastasis is a primary source of morbidity and mortality in cancer. Adrenomedullin (AM) is a multifunctional peptide regulated by receptor activity-modifying proteins (RAMPs). We previously reported that the AM-RAMP2 system is involved in tumor angiogenesis, but the function of the AM-RAMP3 system remains largely unknown. Here, we investigated the actions of the AM-RAMP2 and 3 systems in the tumor microenvironment and their impact on metastasis. PAN02 pancreatic cancer cells were injected into the spleens of mice, leading to spontaneous liver metastasis. Tumor metastasis was enhanced in vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-). By contrast, metastasis was suppressed in RAMP3-/- mice, where the number of podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) was reduced in the periphery of tumors at metastatic sites. Because PDPN-positive CAFs are a hallmark of tumor malignancy, we assessed the regulation of PDPN and found that Src/Cas/PDPN signaling is mediated by RAMP3. In fact, RAMP3 deficiency CAFs suppressed migration, proliferation, and metastasis in co-cultures with tumor cells in vitro and in vivo. Moreover, the activation of RAMP2 in RAMP3-/- mice suppressed both tumor growth and metastasis. Based on these results, we suggest that the upregulation of PDPN in DI-E-RAMP2-/- mice increases malignancy, while the downregulation of PDPN in RAMP3-/- mice reduces it. Selective activation of RAMP2 and inhibition of RAMP3 would therefore be expected to suppress tumor metastasis. This study provides the first evidence that understanding and targeting to AM-RAMP systems could contribute to the development of novel therapeutics against metastasis.

Rapid SERS-based recognition of cell secretome on the folic acid-functionalized gold gratings.

Nowadays, functionalization of the plasmon-supported nanostructured surface is considered as a powerful tool for tumour cell recognition. In this study, the SERS on a surface plasmon polariton-supported gold grating functionalized with folic acid was used to demonstrate an unpretentious recognition of melanoma-associated fibroblasts. Using cultivation media conditioned by different cells, we were able to detect reproducible differences in the secretome of melanoma-associated and normal control fibroblasts. The homogeneous distribution of plasmon energy along the grating surface was proved to provide excellent SERS signal reproducibility, while, to increase the affinity of (bio)molecules to SERS substrate, folic acid molecules were covalently grafted to the gold gratings. As proof of concept, fibroblasts were cultured in vitro, and culture media from the normal and tumour-associated lines were collected and analysed with our proposed SERS substrates. Identifying individual peaks of the Raman spectra as well as comparing their relative intensities, we showed that the proposed functional SERS platform can recognise the melanoma-associated cells without the need for further statistical spectral evaluation directly. We also demonstrated that the SERS chip created provided a stable SERS signal over a period of 90 days without loss of sensitivity. Graphical abstract.

A Multicenter Study of the Prognostic Value of Desmoplastic Reaction Categorization in Stage II Colorectal Cancer.

Highly accurate risk assessment of recurrence may improve adjuvant treatment practice in stage II colorectal cancer (CRC), which lacks definite prognostic factors. Recent studies indicate the importance of stroma in determining cancer behavior, although there are few histopathologic criteria for its evaluation. A pathology review of 679 stage II CRC patients (1980-2005) was conducted at an institution. Desmoplastic reaction (DR) results were classified as mature, intermediate, or immature depending on the presence of hyalinized collagen bundles and myxoid stroma observed at the extramural desmoplastic front on hematoxylin-eosin-stained slides. Pathologically, 430, 180, and 69 tumors were classified into the mature, intermediate, and immature groups, respectively. On the basis of the DR results, 5-year recurrence rate was found to have a wide range of 9.1% to 30.7%; 5-year relapse-free survival (RFS) rates were highest in the mature group (85.2%), followed by the intermediate (77.1%), and immature (60.9%) groups. Multivariate analyses revealed an independent effect of DR pattern on RFS. In addition, 446 patients treated at 4 independent institutions (2007-2008) were examined as a second cohort for result validation, revealing an adverse prognostic impact of unfavorable DR and identifying DR categorization as an independent prognostic factor. In both cohorts, Harrell's concordance index for RFS was higher than the other conventional factors in the DR including T stage. Categorizing DR pattern based on the histologic products of fibroblasts at the desmoplastic front help elucidate their important biological role in cancer development, thus providing clinically useful prognostic information regarding stage II CRC.

Increased expression of cancer-associated fibroblast markers at the invasive front and its association with tumor-stroma ratio in colorectal cancer.

BACKGROUND: The tumor microenvironment has a critical role in regulating cancer cell behavior. Tumors with high stromal content are associated with poor patient outcome. The tumor-stroma ratio (TSR) identifies colorectal cancers (CRC) with poor patient prognosis based on hematoxylin & eosin stained sections. The desmoplastic reaction consists to a great extent of cancer-associated fibroblasts (CAFs) of which different subtypes are known. The aim of this study is to investigate and quantify CAFs present in the tumor stroma of CRC stratified by the TSR to possibly add prognostic significance to the TSR. METHODS: The expression of established CAF markers was compared between stroma-low and stroma-high tumors using transcriptomic data of 71 stage I - III CRC. Based on literature, fibroblast and stromal markers were selected to perform multiplex immunofluorescent staining on formalin fixed, paraffin-embedded tumor sections of patients diagnosed with stage III colon cancer. Antibodies against the following markers were used: αSMA, PDGFR -ß, FAP, FSP1 and the stromal markers CD45 and CD31 as reference. The markers were subsequently quantified in the stroma using the Vectra imaging microscope. RESULTS: The transcriptomic data showed that all CAF markers except one were higher expressed in stroma-high compared to stroma-low tumors. Histologically, stroma-high tumors showed a decreased number of FSP1+/CD45+ cells and a trend of an increased expression of FAP compared to stroma-low tumors. FAP was higher expressed at the invasive part compared to the tumor center in both stroma-high and stroma-low tumors. CONCLUSIONS: The increased expression of FAP at the invasive part and in stroma-high tumors might contribute to the invasive behavior of cancer cells. Future functional experiments should investigate the contribution of FAP to cancer cell invasion. Combining the quantity of the stroma as defined by the TSR with the activity level of CAFs using the expression of FAP may result in an expanded stroma-based tool for patient stratification.

Differences of tumor microenvironment between stage I lepidic-positive and lepidic-negative lung adenocarcinomas.

OBJECTIVE: Lepidic growth is a noninvasive component of lung adenocarcinoma. Many adenocarcinoma cases contain coexistent lepidic and nonlepidic (invasive) components (lepidic-growth positive [Lep+] adenocarcinoma); however, some cases comprise only nonlepidic components (lepidic-growth negative [Lep-] adenocarcinoma). The aim of this study was to investigate the biological differences between the invasive components of Lep+ and Lep- adenocarcinoma. METHODS: We investigated the clinicopathologic characteristics of 232 adenocarcinomas (116 size-matched tumor pairs from Lep+ and Lep- adenocarcinomas). We then evaluated the cancer cell-specific expression levels of cancer stem cell, hypoxia, and invasion molecules in these lesions. The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts and CD204-positive tumor-associated macrophages, was also analyzed. RESULTS: Among cases with size-matched invasive components, significant differences were shown in total tumor size and predominant subtype in invasive component between Lep+ and Lep- adenocarcinomas. The expression levels of hypoxia-related molecules were significantly lower in Lep+ adenocarcinomas (glucose transporter 1: 0 vs 10, P < .01; carbonic anhydrase IX: 0 vs 0 [mean, 4.7 vs 14.1], P = .01). The number of podoplanin-positive cancer-associated fibroblasts and CD204-positive tumor-associated macrophages was significantly lower in Lep+ adenocarcinomas (podoplanin-positive cancer-associated fibroblasts: 0 vs 0 [mean: 1.6 vs 11.6], P < .01; CD204-positive tumor-associated macrophages: 8.7 vs 24.7, P < .01). CONCLUSIONS: Our results indicated that lower cancer cell-specific expression levels of hypoxia markers and a smaller number of tumor-promoting stromal cells in invasive component were characteristic features of Lep+ adenocarcinomas.

Designer hydrogels: Shedding light on the physical chemistry of the pancreatic cancer microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer mortality in the United States, with a 5-year survival of ∼8%. PDAC is characterized by a dense and hypo-vascularized stroma consisting of proliferating cancer cells, cancer-associated fibroblasts, macrophages and immune cells, as well as excess matrices including collagens, fibronectin, and hyaluronic acid. In addition, PDAC has increased interstitial pressures and a hypoxic/acidic tumor microenvironment (TME) that impedes drug delivery and blocks cancer-directed immune mechanisms. In spite of increasing options in targeted therapy, PDAC has mostly remained treatment recalcitrant. Owing to its critical roles on governing PDAC progression and treatment outcome, TME and its interplay with the cancer cells are increasingly studied. In particular, three-dimensional (3D) hydrogels derived from or inspired by components in the TME are progressively developed. When properly designed, these hydrogels (e.g., Matrigel, collagen gel, hyaluronic acid-based, and semi-synthetic hydrogels) can provide pathophysiologically relevant compositions, conditions, and contexts for supporting PDAC cell fate processes. This review summarizes recent efforts in using 3D hydrogels for fundamental studies on cell-matrix or cell-cell interactions in PDAC.

Differential distribution and enrichment of non-coding RNAs in exosomes from normal and Cancer-associated fibroblasts in colorectal cancer.

Exosome production from cancer-associated fibroblasts seems to be an important driver of tumor progression. We report the first in-depth biotype characterization of ncRNAs, analyzed by Next Generation Sequencing and Bioinformatics, expressed in established primary human normal and cancer-associated fibroblasts (CAFs) from cancer and normal mucosa tissues from 9 colorectal cancer patients, and/or packaged in their derived exosomes. Differential representation and enrichment analyses based on these ncRNAs revealed a significant number of differences between the ncRNA content of exosomes and the expression patterns of the normal and cancer-associated fibroblast cells. ncRNA regulatory elements are specifically packaged in CAF-derived exosomes, supporting a specific cross-talk between CAFs and colon cancer cells and/or other stromal cells, mediated by exosomes. These sncRNAs are potential biomarkers present in cancer-associated fibroblast-derived exosomes, which should thereby contribute to developing new non-invasive diagnostic, prognostic and predictive methods for clinical applications in management of cancer patients.

Three-Dimensional Cell Culture Conditions Affect the Proteome of Cancer-Associated Fibroblasts.

In vitro cell culture systems are an invaluable tool for cell biological research to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns about the reliability and conferrability of the obtained results. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, for example, in pharmacological screens. Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigel amenable to stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAF) on 3D culture conditions. Both, control cells and CAF, change their proteomic composition based on the culture conditions. Critically, cell type differences observed in 2D are often not preserved in 3D, which commonly closer resemble phenotypes observed in vivo. Especially, extracellular matrix and plasma membrane proteins are differentially regulated in 2D versus 3D.

EWSR1-SMAD3-rearranged Fibroblastic Tumor: An Emerging Entity in an Increasingly More Complex Group of Fibroblastic/Myofibroblastic Neoplasms.

Three cases of superficial acral fibroblastic spindle cell neoplasms with EWSR1-SMAD3 fusion have been recently reported. Their differential diagnosis is broad, primarily comprising rare tumors from the fibroblastic/myofibroblastic category. The aim of this report is to present 4 new cases of this entity and to discuss the appropriate differential diagnosis. Also, as the ERG antibody seems to be a characteristic marker for these tumors, we analyzed ERG immunostaining characteristics in potential mimics of this entity. All cases in our cohort occurred in women aged 5 to 68 years (mean, 36.5 y). Two were located on the hand, 1 on foot, and the last case arose on the calf. The tumor size ranged from 1 to 1.5 cm in the greatest dimension, with a mean size of 1.2 cm. Except for one recent case, follow-up was available, ranging from 7 to 18 years (mean, 11.7 y), with a recurrence noted in 1 case after 10 years. All tumors were subcutaneous and showed 2 main components. One consisted of bland, spindled cells with elongated nuclei which were round when observed on the cross-section. These cells mostly grew in relatively hypercellular, well-organized, and intersecting fascicles. The second component was prominently hyalinized and paucicellular, but lacked calcifications. Both components showed either a distinct zonation pattern, or they were randomly intermingled with each other. In all 3 analyzable tumors, next-generation sequencing showed EWSR1-SMAD3 gene fusion in each case. By fluorescence in situ hybridization, one tested case also revealed unbalanced rearrangement of the EWSR1 gene. All 4 cases showed strong, diffuse nuclear expression of ERG, whereas none of the mimics stained with this antibody except for weak to moderate staining in calcifying aponeurotic fibromas (9/10 cases). Two tumors showed focal weak to moderate expression of SAT-B2. The 4 herein presented cases further broaden the clinicopathologic spectrum of tumors with EWSR1-SMAD3 gene fusion. They also confirm that they represent a novel entity for which we propose the name EWSR1-SMAD3-rearranged fibroblastic Tumor. Our study also proves that in the context of fibroblastic/myofibroblastic tumors, ERG immunohistochemistry is a relatively specific marker for these neoplasms.

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

Clinical significance of FAP-α on microvessel and lymphatic vessel density in lung squamous cell carcinoma.

AIMS: We aimed to determine whether cancer-associated fibroblasts (CAFs) are associated with microvessel density (MVD) and lymphatic vessel density (LVD) in lung squamous cell carcinoma, as well as their clinical significance in predicting survival. METHODS: 122 patients were enrolled in the study. Samples were obtained on resection at the Department of Thoracic Surgery of the Qingdao Municipal Hospital between January 2011 and December 2014. Immunohistochemistry was used to determine vessel and lymphatic vessel density, and CAF abundance (fibroblast activation protein α (FAP-α) positivity). Statistical analyses were performed on 85 patients to test for correlation of CAF density and other clinicopathological variables with 3-year overall survival (OS) and disease-free survival (DFS). RESULTS: High stromal CAF abundance significantly correlated with increased MVD and LVD in lung squamous cell carcinoma (p<0.05). χ2 test revealed a significant association of CAF density with lymph node metastasis. Cox proportional hazards model showed that both higher CAF density and lymph node metastasis negatively correlate with survival. CAF density or lymph node status can be used as an independent prognostic factor to predict 3-year OS and DFS. CONCLUSIONS: CAF density, identified by FAP-α staining pattern, should be considered as a novel biomarker for disease prognosis in patients with lung squamous cell carcinoma.

Analysis of cancer-associated fibroblasts and the epithelial-mesenchymal transition in cutaneous basal cell carcinoma, squamous cell carcinoma, and malignant melanoma.

Activated cancer-associated fibroblasts (CAFs) and fibroblasts that have undergone the epithelial-mesenchymal transition (EMT) in cancer stroma contribute to tumor progression and metastasis. However, no reports have investigated the CAF phenotype and its clinicopathological relevance in cutaneous malignant tumors, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and malignant melanoma (MM). Here, we investigated the CAF phenotype in cutaneous malignant tumors based on their histology and immunohistochemical expression of CAF-related markers, including adipocyte enhancer-binding protein 1 (AEBP1), podoplanin, platelet-derived growth factor receptor α (PDGFRα), PDGFRß, fibroblast activating protein (FAP), CD10, S100A4, α-smooth muscle actin (α-SMA), and EMT-related markers (Zeb1, Slug, and Twist). In addition, we assessed the role of the CAF phenotype in cutaneous malignant cancers using hierarchical cluster analysis. Consequently, 3 subgroups were stratified based on the expression pattern of CAF- and EMT-related markers. Subgroup 1 was characterized by low expression of AEBP1, PDGFRα, PDGFRß, FAP and Slug, whereas subgroup 2 was closely associated with high expression of PDGFRß, S100A4 and Twist. In addition, high expression levels of podoplanin, PDGFRß, CD10, S100A4, α-SMA, Zeb1, Slug and Twist were observed in subgroup 3. High expression of CD10 was commonly found in all 3 subgroups. These subgroups were correlated with histologic subtypes, that is, subgroup 1, MM; subgroup 2, BCC; and subgroup 3, SCC. We suggest that the expression pattern of CAF- and EMT-related proteins plays crucial roles in the progression of BCC, SCC, and MM.