Presence Of Cancer-associated Fibroblasts Research Articles

Abstract B107: Spatial transcriptomics analysis of PanIN reveals loss of pro-inflammatory signaling and the presence of cancer-associated fibroblasts

Abstract Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion that potentially progresses into pancreatic ductal adenocarcinoma (PDAC). Since PanIN diagnosis is restricted to formalin-fixed and paraffin-embedded (FFPE) samples, little is known about PanIN interactions with the surrounding microenvironment due to the technical limitations to perform genome-wide single-cell analysis. We developed whole transcriptome spatial technology for FFPE to provide the ideal tool to profile PanINs in their microenvironment. We performed FFPE spatial transcriptomics (ST) using the Visium (10x Genomics) approach in a cohort of 14 PanIN lesions (9 low-grade and 5 high-grade). To overcome experimental limitations, we developed an artificial intelligence method to integrate imaging, spatial transcriptomics, and single-cell RNA-seq (scRNA-seq) analyses. Imaging analysis of the same stained sections used for ST was applied before gene expression exploration. Using machine learning to annotate cell types, we were able to deconvolve and quantify the cell types per ST spot using cell morphology instead of a scRNA-seq gene expression reference. Subsequently, we applied transfer learning to a scRNA-seq PDAC atlas that was built from multiple published datasets. The whole transcriptome ST findings were confirmed using targeted single-cell transcriptomics (Xenium, 10x Genomics) and proteomics (imaging mass cytometry, IMC). The analysis revealed that PanINs already exhibit molecular and cellular features of PDAC, including expression of the PDAC classical subtype signature and the presence of the same cancer-associated fibroblast (CAF) subtypes enriched in invasive carcinoma stages. The CAF subtypes, including the recently described antigen-presenting CAFs, were confirmed at single-cell resolution using Xenium and IMC. Integrating multi-omics data with a PDAC scRNA-seq atlas through transfer learning, revealed that an inflammatory pattern gradually decreases during PanIN progression, accompanied by a compensatory increase in proliferation pathways in the PanIN cells relative to normal duct cells. This trend between inflammation and proliferation was confirmed specifically in PanIN cells using Xenium. Our experimental and computational approach provides valuable insights into the dynamics of pancreatic tumor progression from PanIN lesions using innovative methods to provide a unique spatial multi-omics reference of PanINs. Citation Format: Alexander T.F. Bell, Jacob T. Mitchell, Ashley L. Kiemen, Melissa Lyman, Kohei Fujikura, Jae W. Lee, Erin Coyne, Sarah M. Shin, Pei-Hsun Wu, Jacquelyn W. Zimmerman, Denis Wirtz, Won J. Ho, Neeha Zaidi, Elizabeth Thompson, Elizabeth M. Jaffee, Laura D. Wood, Elana J. Fertig, Luciane T. Kagohara. Spatial transcriptomics analysis of PanIN reveals loss of pro-inflammatory signaling and the presence of cancer-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B107.

α-SMA+ cancer-associated fibroblasts increased tumor enhancement ratio on contrast-enhanced multidetector-row computed tomography in stages I-III colon cancer.

Our prior research revealed that the tumor enhancement ratio (TER) on triphasic abdominal contrast-enhanced MDCT (CE-MDCT) scans was a prognostic factor for patients with stages I-III colon cancer. Building upon this finding, the present study aims to investigate the proteomic changes in colon cancer patients with varying TER values. TER was analyzed on preoperative triphasic CE-MDCT scans of 160 stages I-III colon cancer patients. The survival outcomes of those in the low-TER and high-TER groups were compared. Proteomic analysis on colon cancer tissues was performed by mass spectrometry (MS) and verified by immune-histological chemistry (IHC) assays. In vivo, mouse xenograft models were employed to test the function of target proteins identified through the MS. CE-MDCT scans were conducted on mice xenografts, and the TER values were compared. Patients in the high-TER group had a significantly worse prognosis than those in the low-TER group. Proteomic analysis of colon cancer tissues revealed 153 differentially expressed proteins between the two groups. A correlation between TER and the abundance of α-SMA protein in tumor tissue was observed. IHC assays further confirmed that α-SMA protein expression was significantly increased in high-TER colon cancer, predominantly in cancer-associated fibroblasts (CAFs) within the cancer stroma. Moreover, CAFs promoted the growth of CRC xenografts in vivo and increased TER. Our study identified the distinct protein changes in colon cancer with low and high TER for the first time. The presence of CAFs may promote the growth of colon cancer and contribute to an increased TER.

A generalized AI method for pathology cancer diagnosis and prognosis prediction based on transfer learning and hierarchical split

Objective. This study aims to propose a generalized AI method for pathology cancer diagnosis and prognosis prediction based on transfer learning and hierarchical split. Approach. We present a neural network framework for cancer diagnosis and prognosis prediction in pathological images. To enhance the network’s depth and width, we employ a hierarchical split block (HS-Block) to create an AI-aided diagnosis system suitable for semi-supervised clinical settings with limited labeled samples and cross-domain tasks. By incorporating a lightweight convolution unit based on the HS-Block, we improve the feature information extraction capabilities of a regular network (RegNet). Additionally, we integrate a Convolutional Block Attention Module into the first and last convolutions to optimize the extraction of global features and local details. To address limited sample labels, we employ a dual-transfer learning (DTL) mechanism named DTL-HS-Regnet, enabling semi-supervised learning in clinical settings. Main results. Our proposed DTL-HS-Regnet model outperforms other advanced deep-learning models in three different types of cancer diagnosis tasks. It demonstrates superior feature extraction ability, achieving an average sensitivity, specificity, accuracy, and F1 score of 0.9987, 1.0000, 1.0000 and 0.9992, respectively. Furthermore, we evaluate the model’s capability to directly extract prognosis prediction information from pathological images by constructing patient cohorts. The results show that the correlation between DTL-HS-Regnet predictions and the presence of cancer-associated fibroblasts is comparable to that of pathologists. Significance. Our proposed AI method offers a generalized approach for cancer diagnosis and prognosis prediction in pathology. The outstanding performance of the DTL-HS-Regnet model demonstrates its potential for improving current practices in image digital pathology, expanding the boundaries of cancer treatment in two critical areas.

Identification of Cancer-Associated Fibroblasts in Metastatic Lymph Nodes of Colorectal Carcinoma.

Cancer-associated fibroblasts (CAF) are a heterogeneous group of tumor microenvironment cells that are barely studied in metastatic lymph nodes. The presence of CAF in regional metastases of colorectal cancer was assessed by using SMA, PDGFRb, and POD markers; the obtained subpopulations were compared with the primary tumor. A total of 26 cases of colon adenocarcinoma with metastases to regional lymph nodes were studied. Duplex immunohistochemical detection (POD+SMA and PDGFRb+SMA) was carried out by the immunohistochemical method. In most cases, POD was absent in metastases (65.4%) and PDGFRb was present (88.5%). The POD and PDGFRb staining in the invasive edge of the tumor did not correlate with metastasis. Attention was drawn to the absence of POD and PDGFRb reactions in a blood vessel embolus, as well as to negative PDGFRb in metastasis in the presence of pronounced PDGFRb in the primary tumor in patients after neoadjuvant therapy.

Asocijacija fibroplasta povezanih sa karcinomom sa kliničko-patološkim parametrima oralnog karcinoma skvamoznih ćelija - imunohistemijska studija

Introduction: Cancer-associated fibroblasts (CAFs) from the tumor microenvironment (TME) play an important role in the progression, recurrence and poor prognosis of OSCC. The aim: To determine the density of CAFs in the tumorstroma (TS) of OSCC, and to determine the association of the density of CAFs with clinico-pathological parameters and disease relapse in patients with OSCC. Materials and methods: Immunohistochemical staining of tissue samples from primary OSCC was performed in 45 patients operated at the University Clinic for Maxillofacial Surgery, in Skopje, North Macedonia. A mouse primary monoclonal antibody: anti-a-SMA was used to visualize CAFs. The density of CAFs aSMA+ was grouped into 4 grades, and the obtained data were statistically analyzed. Results: The presence of CAFs was not determined in tissue samples from all patients with OSCC. There is a significant association of the density of CAFs in the primary tumor with T, N and TNM-status, respectively (p=0.0006, p=0.0255, p=0.0164). The Difference test determined that only in case of disease relapse in the form of local recurrence, the percentage representation of patients with CAFs present was significantly higher compared to patients who did not have CAFs (p=0.0001). Conclusions: These findings are suggestive of the role of CAFs in disease progression, but are insufficient to be included as a parameter in a standardized histopathological finding.

Fibroblasts and macrophages cooperate to create a pro-tumorigenic and immune resistant environment via activation of TGF-β/IL-6 pathway in neuroblastoma

ABSTRACT Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors, but how they cooperate is not well understood. Here, we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-β1, MCP-1, IL-6, IL-8, and IL-4) but not of anti-tumorigenic cytokines (TNF-α, IL-12) by MN or MSC, while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-β1/IL-6 pathway where TGF-β1 stimulated the expression of IL-6 in NB cells and MSC, promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer.

MEG3 Expression Indicates Lymph Node Metastasis and Presence of Cancer-Associated Fibroblasts in Papillary Thyroid Cancer.

Papillary thyroid cancer is the most common endocrine malignancy, occurring at an incidence rate of 12.9 per 100,000 in the US adult population. While the overall 10-year survival of PTC nears 95%, the presence of lymph node metastasis (LNM) or capsular invasion indicates the need for extensive neck dissection with possible adjuvant radioactive iodine therapy. While imaging modalities such as ultrasound and CT are currently in use for the detection of suspicious cervical lymph nodes, their sensitivities for tumor-positive nodes are low. Therefore, advancements in preoperative detection of LNM may optimize the surgical and medical management of patients with thyroid cancer. To this end, we analyzed bulk RNA-sequencing datasets to identify candidate markers highly predictive of LNM. We identified MEG3, a long-noncoding RNA previously described as a tumor suppressor when expressed in malignant cells, as highly associated with LNM tissue. Furthermore, the expression of MEG3 was highly predictive of tumor infiltration with cancer-associated fibroblasts, and single-cell RNA-sequencing data revealed the expression of MEG3 was isolated to cancer-associated fibroblasts (CAFs) in the most aggressive form of thyroid cancers. Our findings suggest that MEG3 expression, specifically in CAFs, is highly associated with LNM and may be a driver of aggressive disease.

CGAS-STING signaling encourages immune cell overcoming of fibroblast barricades in pancreatic cancer

Immune checkpoint blockade (ICB) treatment improves the prognosis of several types of solid tumors, however, responsiveness to ICB therapy remains low in pancreatic ductal adenocarcinoma (PDACs), which has a rich tumor microenvironment (TME). The TME is composed of various stromal cells, including cancer-associated fibroblasts (CAFs), which contribute to the establishment of an immunosuppressive microenvironment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an innate immune pathway that results in the upregulation of immune cell recruiting-cytokines and anti-tumor efficacy. In this study, we aimed to investigate the impact of cGAS-STING expression and the presence of CAFs upon immune cell infiltration in PDACs. cGAS and STING co-expressing PDAC cases showed favorable survival, with many cytotoxic CD8 + T cell infiltrations from the stromal component adjacent to the cancer cells toward cancer cells, but not in cGAS-STING signaling defected PDAC cases. The signatures of tumor-restrain CAFs were expressed in tumors with cGAS-STING signaling. Finally, transwell co-culture experiments demonstrated that immune cell infiltration was impeded by the presence of CAFs, but not by activation of cGAS-STING signaling. In conclusion, pro-infiltration signals, such as cGAS-STING, and characterization of CAFs are crucial in defeating CAF barricades and encouraging immune cell infiltration in PDACs.

Background features in the cytology of pancreatic neoplasms.

Cytology is a useful method for diagnosing pancreatic neoplasms. Although endoscopic ultrasound‐guided fine‐needle aspiration has recently become the mainstream method for the diagnosis of pancreatic neoplasms, pancreatic juice and pancreatic duct brushing cytology continue to be useful diagnostic methods for the investigation of pancreatic neoplasms. Diagnoses using pancreatic cytology are primarily based on the features related to tumor cells; however, evaluation of the background features provides important information that could further aid the diagnosis. Pancreatic neoplasms show various histological types, each of which is associated with its own characteristic background features. The necrotic background, desmoplastic stroma, and presence of cancer‐associated fibroblasts are background features of pancreatic ductal adenocarcinoma, a mucinous background is associated with intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, and hyaline globules are observed in solid pseudopapillary neoplasms. However, some background features are associated with more than one histological type of pancreatic neoplasm, highlighting the importance to base a diagnosis on the results of a comprehensive analysis of not only the background features but also the tumor cells. Here, we provide a review of the key background cytological features of pancreatic neoplasms, which can serve as a guide to improve diagnosis and research.

Pro-Inflammatory Cytokines Trigger the Overexpression of Tumour-Related Splice Variant RAC1B in Polarized Colorectal Cells

Simple SummaryTumours are now known to develop more quickly when the tumour cell mass is located in a tissue that shows signs of chronic inflammation. Under such conditions, inflammatory cells from the surrounding tumour microenvironment provide survival signals to which cancer cells respond. We have previously found that some colorectal tumours overexpress the protein RAC1B that sustains tumour cell survival. Here we used a colon mucosa-like in vitro cell model and found that the presence of cancer-associated fibroblasts and pro-inflammatory macrophages stimulated colorectal cells to increase their RAC1B levels. Under these conditions, the secreted survival signals were analysed, and interleukin-6 identified as the main trigger for the increase in RAC1B levels. The results contribute to understand the tumour-promoting effect of inflammation at the molecular level.An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Review: Challenges of In Vitro CAF Modelling in Liver Cancers

Simple SummaryLiver cancer and tumours spreading from other organs to the liver are associated with high death rates. Current treatments include surgical removal of the tumour and chemotherapy. Unfortunately, patients are often re-diagnosed with liver nodules in the years after cessation of the treatment. Therefore, scientists are looking for alternative treatment strategies, and these include targeting the tumour environment. The tumour environment includes the cancer-associated fibroblasts, which could be an interesting target for therapy in combination with current strategies. In this review paper we summarize the current models to investigate the effect of the tumour on the cancer-associated fibroblasts. Not many studies focus on the cancer-associated fibroblasts in non-animal models and this should improve in order to better understand the role of the cancer-associated fibroblasts and to evaluate the potential of cancer-associated fibroblast-directed therapies.Primary and secondary liver cancer are the third cause of death in the world, and as the incidence is increasing, liver cancer represents a global health burden. Current treatment strategies are insufficient to permanently cure patients from this devastating disease, and therefore other approaches are under investigation. The importance of cancer-associated fibroblasts (CAFs) in the tumour microenvironment is evident, and many pre-clinical studies have shown increased tumour aggressiveness in the presence of CAFs. However, it remains unclear how hepatic stellate cells are triggered by the tumour to become CAFs and how the recently described CAF subtypes originate and orchestrate pro-tumoural effects. Specialized in vitro systems will be needed to address these questions. In this review, we present the currently used in vitro models to study CAFs in primary and secondary liver cancer and highlight the trend from using oversimplified 2D culture systems to more complex 3D models. Relatively few studies report on the impact of cancer (sub)types on CAFs and the tumour microenvironment, and most studies investigated the impact of secreted factors due to the nature of the models.

Phenotype transition of fibroblasts incorporated into patient-derived oral carcinoma organoids.

Cancer-associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact-dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast-attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell-clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Compared to traditional 3D co-culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. We developed a robust strategy to study contact-dependent mechanisms underlying tumour-stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC-associated fibroblasts.

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.

Cancer-associated fibroblasts are associated with poor prognosis in solid type of lung adenocarcinoma in a machine learning analysis

Cancer-associated fibroblasts (CAFs) participate in critical processes in the tumor microenvironment, such as extracellular matrix remodeling, reciprocal signaling interactions with cancer cells and crosstalk with infiltrating inflammatory cells. However, the relationships between CAFs and survival are not well known in lung cancer. The aim of this study was to reveal the correlations of CAFs with survival rates, genetic alterations and immune activities. This study reviewed the histological features of 517 patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA) database. We performed gene set enrichment analysis (GSEA), network-based analysis and survival analysis based on CAFs in four histological types of lung adenocarcinoma: acinar, papillary, micropapillary and solid. We found four hallmark gene sets, the epithelial-mesenchymal transition, angiogenesis, hypoxia, and inflammatory response gene sets, that were associated with the presence of CAFs. CAFs were associated with tumor proliferation, elevated memory CD4+T cells and high CD274 (encoding PD-L1) expression. In the pathway analyses, CAFs were related to blood vessel remodeling, matrix organization, negative regulation of apoptosis and transforming growth factor-β signaling. In the survival analysis of each histological type, CAFs were associated with poor prognosis in the solid type. These results may contribute to the development of therapeutic strategies against lung adenocarcinoma cases in which CAFs are present.

Cancer-associated fibroblasts in renal cell carcinoma: implication in prognosis and resistance to anti-angiogenic therapy.

To investigate the role of cancer-associated fibroblasts (CAFs) in clear cell renal cell carcinoma (ccRCC) with respect to tumour aggressiveness, metastasis development, and resistance to anti-angiogenic therapy (vascular endothelial growth factor receptor-tyrosine kinase inhibitors [VEGFR-TKI]). Our study involved tissue samples from three distinct and independent cohorts of patients with ccRCC. The presence of CAFs and tumour lymphangiogenesis was investigated, respectively, by transcriptional signatures and then correlated with tumour development and prognosis. The effect of these CAFs on tumour cell migration and VEGFR-TKI resistance was analysed on co-cultures of ccRCC cells with CAFs. Results from our cohorts and from in silico investigations showed that VEGFR-TKI significantly increase the number of CAFs in tumours. In the same populations of patients with ccRCC, the proportion of intra-tumoral CAFs correlated to shorter disease-free and overall survival. The presence of CAFs was also correlated with lymphangiogenesis and lymph node metastasis. CAFs increased the migration and decreased the VEGFR-TKI-dependent cytotoxic effect of tumour cells. Our results show that VEGFR-TKI promote the development of CAFs, and CAFs favour tumour aggressiveness, metastatic dissemination, and resistance to treatment in ccRCC. CAFs could represent a new therapeutic target to fight resistance to treatment of ccRCC. Targeting CAF and immunotherapies combination are emerging as efficient treatments in many types of solid tumours. Our results highlight their relevance in ccRCC.

The role of α-smooth muscle actin in confirming the microinvasion of laryngeal squamous cell carcinoma

BackgroundThe diagnosis of microinvasive laryngeal squamous cell carcinoma (LSCC) is not always straightforward and sometimes can be very challenge in daily clinical practice. The focus lies in the confirmation of microinvasion. Cancer-associated fibroblasts (CAFs), as the major element of reactive tumor stroma, are believed to participate actively in the growth and invasion of tumor cells. ObjectivesTo evaluate the diagnostic role of α-smooth muscle actin (α-SMA) labelling CAFs in microinvasive LSCC. MethodsA total of 81 laryngeal biopsy specimens were retrieved, including 41 cases of microinvasive LSCC with depth of invasion no more than 3 mm, 20 laryngeal squamous intraepithelial lesion (SIL), and 20 benign pseudoepitheliomatous hyperplasia (PEH). All cases were stained for immunohistochemistry, using antibody against the α-SMA antigen. The correlation between the presence of CAFs in microinvasive LSCC and tumor histological characteristics was investigated. ResultsImmunoreactivity of α-SMA was detected in twenty-nine microinvasive LSCC (29/41, 71%), while no reactivity was observed in laryngeal SIL (0/20, 0%), and rarely in PEH (2/20, 10%). The α-SMA expression pattern in stroma of microinvasive LSCC was significantly different from that of SIL (χ2 = 26.966, p = 0.000) and PEH (χ2 = 19.838, p = 0.000). In addition, there seemed to be a certain correlation between the histological characteristics of microinvasive LSCC and the presence of interstitial CAFs. ConclusionsThis study highlights the practical role of utilizing α-SMA in the pathological diagnosis of microinvasive LSCC, with emphasis on variable histomorphologic features of microinvasive LSCC.

The EBV-Encoded Oncoprotein, LMP1, Recruits and Transforms Fibroblasts via an ERK-MAPK-Dependent Mechanism

Latent membrane protein 1 (LMP1), the major oncoprotein encoded by Epstein–Barr virus (EBV), is expressed at widely variable levels in undifferentiated nasopharyngeal carcinoma (NPC) biopsies, fueling intense debate in the field as to the importance of this oncogenic protein in disease pathogenesis. LMP1-positive NPCs are reportedly more aggressive, and in a similar vein, the presence of cancer-associated fibroblasts (CAFs) surrounding “nests” of tumour cells in NPC serve as indicators of poor prognosis. However, there is currently no evidence linking LMP1 expression and the presence of CAFs in NPC. In this study, we demonstrate the ability of LMP1 to recruit fibroblasts in vitro in an ERK-MAPK-dependent mechanism, along with enhanced viability, invasiveness and transformation to a myofibroblast-like phenotype. Taken together, these findings support a putative role for LMP1 in recruiting CAFs to the tumour microenvironment in NPC, ultimately contributing to metastatic disease.

Tumor Microenvironment in Oral Cancer Following Neoadjuvant Pembrolizumab: Preliminary Analysis of the Histopathologic Findings.

Background: The tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC) is associated with immune suppression, one of the pathways being the programmed death receptor 1 (PD-1) and its ligands (PD-L1/PD-L2). Checkpoint inhibitors of PD-1/PD-L1, like pembrolizumab, have been recently approved for treatment of OSCC. We described the histologic findings in OSCC following neoadjuvant pembrolizumab, including identification of immune-related cell populations and cancer-associated fibroblasts (CAFs).Materials and Methods: Patients with OSCC clinical stages 3 and 4 and a combined PD-L1 score >1 were randomized either to the standard oncologic protocol or to the pembrolizumab arm of MK-3475-689 study for Head and Neck, Lip, and Oral Cavity. The latter were given two standard doses of 200 mg of pembrolizumab, 3 weeks apart, and then underwent surgical oncologic procedure according to the initial stage. Sections from the resection specimens were analyzed for pathological response to pembrolizumab. Various populations of immune-related cells within the tumor microenvironment were characterized by immunohistochemistry, as were the CAFs.Results: Three patients who were randomized to the pembrolizumab study were described. One patient presented with a tongue SCC, the other two had SCC of the mandibular ridge with bony involvement. Only the patient with tongue SCC showed clinical complete response. Microscopically, the tumor was replaced by a granulomatous type of inflammation. Immunohistochemical stains revealed massive T cell rich (CD3+) infiltrate, with approximately equal amounts of CD4+ and CD8+ cells, numerous macrophages of CD68+ and CD163+ phenotypes; no CAFs were identified. The other two patients were regarded as non-responders as at least 50% of the tumor was viable. The tumor microenvironment of these tumors was generally associated with a lesser extent of inflammatory response compared to the tongue tumor, a variable CD4+/CD8+ ratio and presence of CAFs. Neither T regulatory cells (FOXP3+) nor natural killer cells (CD56+, CD57+) were identified in any of the cases.Conclusion: We showed that characterizing the specific populations of immune-related cells and CAFs after treatment with pembrolizumab, may add to our understanding of the tumor-TME interactions in this setting. These findings should be investigated in future studies on a larger number of patients.

Abstract PO028: Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation

Abstract The tumour microenvironment (TME), consisting of stromal cell types including fibroblasts, endothelial, immune and mesenchymal stromal cells (MSCs), is an important mediator of breast cancer (BCa) progression. BCa cells regulate the composition of the TME by secreting a myriad of factors; however, mechanisms governing the dynamic reciprocity between BCa and TME components are largely unknown. Cancer-associated fibroblasts (CAFs) are a major stromal component in many BCa and have been shown to be activated by BCa cells. MSCs recruited to the TME are an important source of CAFs in many cancers; therefore, characterizing the factors involved in MSC recruitment could provide additional avenues for therapeutic intervention by disrupting the TME in BCa. NODAL, an embryonic morphogen belonging to the TGF-beta superfamily, is clinically associated with BCa progression, and has been shown to sustain BCa stem cells and to promote tumour growth, metastasis, and vascularization. Herein, we report that NODAL expression positively correlates with the presence of CAFs in the stroma of triple-negative human BCa tissues, as determined by NODAL and a-smooth muscle Actin (SMA) staining. Further, our in vitro studies show that NODAL directly induces primary fibroblast activation and chemotaxis. Proteomic analysis of the conditioned media of NODAL-overexpressing MDA-MB-231 and SUM149 BCa cells by mass spectrometry reveal that NODAL reprograms BCa secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), growth factors (e.g. PDGFRA) and cytokines (e.g. IL-6), and that the NODAL-regulated secretome, but not NODAL directly, impacts the ability of BCa cells to affect MSC chemotaxis. We provide evidence that IL-6 promotes MSC chemotaxis, and further discuss the context-dependent effects of NODAL in BCa cell lines. Collectively, our results suggest that factors present in NODAL-regulated secretomes may induce MSC recruitment to the breast TME, where they may contribute to CAF population, therefore demonstrating a hitherto unappreciated role of NODAL as a dynamic regulator of breast TME components. Citation Format: Dylan Dieters-Castator, Paola M. Dantonio, Matt Piaseczny, Guihua Zhang, Jiahui Liu, Miljan Kuljanin, Stephen Sherman, Michael Jewer, Katherine Quesnel, Eun-Young Kang, Martin Köbel, Gabrielle M. Siegers, Andrew Leask, David Hess, Gilles Lajoie, Lynne-Marie Postovit. Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO028.