Glutamine deficiency drives transforming growth factor-ß signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-ß (TGF-ß)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-ß-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-ß-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-ß-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-ß-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-ß signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Cancer epithelia-derived mitochondrial DNA is a targetable initiator of a paracrine signaling loop that confers taxane resistance.

Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity.

ELF3 mediates IL-1α induced differentiation of mesenchymal stem cells to inflammatory iCAFs.

Stromal cells in the tumor microenvironment regulate the immune landscape and tumor progression. Yet, the ontogeny and heterogeneity of reactive stromal cells within tumors is not well understood. Carcinoma-associated fibroblasts exhibiting an inflammatory phenotype (iCAFs) have been identified within multiple cancers; however, mechanisms that lead to their recruitment and differentiation also remain undefined. Targeting these mechanisms therapeutically may be important in managing cancer progression. Here, we identify the ELF3 transcription factor as the canonical mediator of IL-1α-induced differentiation of prostate mesenchymal stem cells to an iCAF phenotype, typical of the tumor microenvironment. Furthermore, IL-1α-induced iCAFs were subsequently refractive to TGF-ß1 induced trans-differentiation to a myofibroblast phenotype (myCAF), another key carcinoma-associated fibroblast subtype typical of reactive stroma in cancer. Restricted trans-differentiation was associated with phosphorylation of the YAP protein, indicating that interplay between ELF3 action and activation of the Hippo pathway are critical for restricting trans-differentiation of iCAFs. Together, these data show that the IL-1α/ELF3/YAP pathways are coordinate for regulating inflammatory carcinoma-associated fibroblast differentiation.

Metabolic reprogramming of carcinoma-associated fibroblasts and its impact on metabolic heterogeneity of tumors.

Tumor metabolism is characterized with up-regulated glucose uptake and glycolytic rate of tumor cells as the source of ATP and tumors growth, and regulated by a poorly defined combination of cell-intrinsic and extrinsic factors. Metabolic heterogeneity of human tumors is dependent upon the mutational status of specific oncogenes and influenced by tumor microenvironment. Carcinoma-associated fibroblasts (CAFs) adapt in a dynamic manner to the metabolic needs of cancer cells, associated with tumorigenesis and resistance to treatments. Importantly, CAFs could directly "feed" cancer cells essential nutrients and energy-rich metabolites, including lactate, ketone bodies, fatty acids, glutamine, and other amino acids through the induction of autophagy in a host-parasite pattern, to contribute to tumor growth and metastasis. To define the reciprocal metabolic interplay between CAFs and cancer cells will provide a better understanding of molecular mechanisms by which the treatment resistance occurs,and aid in the rational design of metabolism-based approaches to enhance the efficacy of immunotherapy.

Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-metastatic niche in the lung through activating fibroblasts.

OBJECTIVES: Carcinoma-associated fibroblasts (CAFs) have been known to promote cancer progression by modifying the primary tumor microenvironment. We aimed to elucidate the intercellular communication between CAFs and secondary organs in salivary adenoid cystic carcinoma (SACC) metastasis. METHODS: Pre-metastatic and metastatic animal models of SACC were established using extracellular vesicles (EVs) from CAFs and SACC cells. Lung fibroblasts (LFs) were treated with EVs and their transcriptomic alterations were identified by RNA sequencing. ITRAQ were performed to analyze EV cargos. TC I-15 was used to inhibit EV uptake by LFs and SACC lung metastasis in vivo. RESULTS: Here, we show that CAF EVs induced lung pre-metastatic niche formation in mice and consequently increased SACC lung metastasis. The pre-metastatic niche induced by CAF EVs was different from that induced by SACC EVs. CAF EVs presented a great ability for matrix remodeling and periostin is a potential biomarker characterizing the CAF EV-induced pre-metastatic niche. We found that lung fibroblast activation promoted by CAF EVs was a critical event at the pre-metastatic niche. Integrin α2ß1 mediated CAF EV uptake by lung fibroblasts, and its blockage by TC I-15 prevented lung pre-metastatic niche formation and subsequent metastasis. Plasma EV integrin ß1 was considerably upregulated in the mice bearing xenografts with high risk of lung metastasis. CONCLUSIONS: We demonstrated that CAF EVs participated in the pre-metastatic niche formation in the lung. Plasma EV integrin ß1 might be a promising biomarker to predict SACC metastasis at an early stage. An integrated strategy targeting both tumor and stromal cells is necessary to prevent SACC metastasis.

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

Myofibroblasts in oral potentially malignant disorders: Is it related to malignant transformation?

In oral cancer, acquisition of α-smooth muscle actin (α-SMA)-positive fibroblasts, known as myofibroblasts or carcinoma-associated fibroblasts (CAF), is an important event for progression and metastasis. However, the contribution of myofibroblasts in oral potentially malignant disorders (OPMD) remains controversial. This systematic review provides evidence that immunodetection of myofibroblasts may identify oral submucous fibrosis (OSMF) with high risk of malignant transformation, but does not represent an auxiliary tool to predict the malignant potential of leukoplakia and erythroplakia, the most common OPMD.

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted Photoimmunotherapy To Enhance Cytotoxic T Cell Infiltration and Tumor Control.

Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle-based photoimmunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier, and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photoirradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor-bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C-X-C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer.

Mesenchymal Stem Cells are Recruited and Activated into Carcinoma-Associated Fibroblasts by Prostate Cancer Microenvironment-Derived TGF-ß1.

Tumor stromal cells can supply appropriate signals that may develop aggressive phenotypes of carcinoma cells and establish a complex scenario which culminates in metastasis. Recent works proposed that bone marrow-derived mesenchymal stem cells (MSC) are recruited to primary tumors. However, the exact functions of these cells in the tumor microenvironment are not well characterized, as it is reported that MSC can either promote or inhibit tumor progression. In the present study, we aim at investigating the signaling molecules which regulate the interplay between MSC, prostate carcinoma (PCa) cells and two important cellular types constituting the tumor-associated stroma, macrophages and fibroblasts, during their progression toward malignancy. We identified TGF-ß1 as a crucial molecule able to attract MSC recruitment both to PCa cells as well as to tumor stroma components. Moreover, PCa- and tumor stroma-secreted TGF-ß1 is important to induce MSC transdifferentiation into carcinoma-associated fibroblast (CAF)-like cells. Consequently, the CAF-like phenotype acquired by MSC is central to promote tumor progression related effects. Thus, tumor-educated MSC enhance PCa invasiveness compared to nonactivated MSC. Additionally, differing from normal MSC, CAF-like MSC perform vascular mimicry and recruit monocytes, which can be further polarized to M2 macrophages within the PCa environment. Our findings indicate a prominent role for TGF-ß1 in MSC mobilization and activation strengthened by the fact that the blockade of TGF-ß1 signaling impairs MSC promotion of PCa progression. Stem Cells 2016;34:2536-2547.

Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways.

Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data.

Analysis of gene expression changes associated with human carcinoma-associated fibroblasts in non-small cell lung carcinoma.

BACKGROUND: This study aimed to investigate the gene expression changes associated with carcinoma-associated fibroblasts (CAFs) involving in non-small cell lung carcinoma (NSCLC). METHODS: We downloaded the GEO series GSE22862, which contained matched gene expression values for 15 CAF and normal fibroblasts samples, and series GSE27289 containing SNP genotyping for four matched NSCLC samples. The differentially expressed genes in CAF samples were identified using the limma package in R. Then we performed gene ontology (GO) and pathway enrichment analysis and protein-protein interaction (PPI) network construction using the identified DEGs. Moreover, aberrant cell fraction, ploidy, allele-specific copy number, and loss of heterozygosity (LOH) within CAF cells were analyzed using the allele-specific copy number analysis. RESULTS: We obtained 545 differentially expressed genes between CAF and normal fibroblasts samples. The up-regulated genes are mainly involved in GO terms such as positive regulation of cell migration and extracellular region, while the down-regulated genes participate in the lung development and extracellular region. Multiple genes including bone morphogenetic protein 4 (BMP4) and transforming growth factor, beta 3 (TGFB3) are involved in the TGF-ß signaling pathway. Genes including BMP4, TGFBI and matrix Gla protein (MGP) were hub genes. Moreover, no LOH event for BMP4 and MGP was found, that for sphingosine kinase 1 (SPHK1) was 70%, and for TGFBI was 40%. CONCLUSION: Our data suggested that BMP4, MGP, TGFBI, and SPHK1 may be important in CAFs-associated NSCLC, and the abnormal expression and high LOH frequency of them may be used as the diagnosis targets of CAFs in NSCLC.

The impact of tumor stroma on drug response in breast cancer.

In the last two decades the breast cancer mortality rate has steadily declined, in part, due to the availability of better treatment options. However, drug resistance still remains a major challenge. Resistance can be an inherent feature of breast cancer cells, but can also arise from the tumor microenvironment. This review aims to focus on the modulatory effect of the tumor microenvironment on the differing response of breast cancer subtypes to targeted drugs and chemotherapy.

Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS: Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-ß measured by ELISA. RESULTS: MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-ß concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS: Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer.

Carcinoma-associated fibroblasts provide operational flexibility in metastasis.

Malignant cancer cells do not act as lone wolves to achieve metastasis, as they exist within a complex ecosystem consisting of an extracellular matrix scaffold populated by carcinoma-associated fibroblasts (CAFs), endothelial cells and immune cells. We recognize local (primary tumor) and distant ecosystems (metastasis). CAFs, also termed myofibroblasts, may have other functions in the primary tumor versus the metastasis. Cellular origin and tumor heterogeneity lead to the expression of specific markers. The molecular characteristics of a CAF remain in evolution since CAFs show operational flexibility. CAFs respond dynamically to a cancer cell's fluctuating demands by shifting profitable signals necessary in metastasis. Local, tissue-resident fibroblasts and mesenchymal stem cells (MSCs) coming from reservoir sites such as bone marrow and adipose tissue are the main progenitor cells of CAFs. CAFs may induce awakening from metastatic dormancy, a major cause of cancer-specific death. Cancer management protocols influence CAF precursor recruitment and CAF activation. Since CAF signatures represent early changes in metastasis, including formation of pre-metastatic niches, we discuss whether liquid biopsies, including exosomes, may detect and monitor CAF reactions allowing optimized prognosis of cancer patients.

Pharmacokinetics and toxicology of a fibroblast activation protein (FAP)-activated prodrug in murine xenograft models of human cancer.

BACKGROUND: As carcinoma progresses, the stroma undergoes a variety of phenotypic changes, including the presence of carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). FAP is a post-prolyl endopeptidase whose expression in a healthy adult is largely restricted to the cancer-associated stroma. FAP-targeted prodrugs with a 100-fold greater therapeutic window over the parent compound were previously generated. METHODS: Prodrugs and non-cleavable controls were incubated in the presence of FAP. Plasma and tumor half-lives (t1/2) of the full-length and active forms of the prodrugs were determined using LCMS. Biodistribution studies of prodrug activation were performed. Histopathological analysis of tissues from treated animals were compared to vehicle-treated controls. Toxicity and efficacy studies were performed in human breast (MDA-MB-231 and MCF-7) and prostate (LNCaP) cancer xenografts models. RESULTS: These FAP-activated prodrugs have a significantly slower clearance from tumor tissue than the circulation (∼12 vs. ∼4.5 hr). Micromolar concentrations of active drug persist in the tumor. Active drug is detected in non-target tissues; however, histopathologic evaluation reveals no evidence of drug-induced toxicity. A FAP-activated prodrug (ERGETGP-S12ADT) inhibits tumor growth in multiple human breast and prostate cancer xenograft models. The anti-tumor effect is comparable to that observed with docetaxel, but results in significantly less toxicity. CONCLUSION: FAP-activated prodrugs are a viable strategy for the management of prostate and other cancers. These prodrugs exhibit less toxicity than a commonly used chemotherapeutic agent. Further refinement of the FAP cleavage site for greater specificity may reduce prodrug activation in non-target tissues and enhance clinical benefit.

Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis.

Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our in vitro data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events.Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFß: transforming growth factor beta.

Spatial analysis of stromal signatures identifies invasive front carcinoma-associated fibroblasts as suppressors of anti-tumor immune response in esophageal cancer.

BACKGROUND: Increasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC). METHODS: Herein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages. RESULTS: We demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy. CONCLUSIONS: Our newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Exosomal miR-146b-5p derived from cancer-associated fibroblasts promotes progression of oral squamous cell carcinoma by downregulating HIPK3.

OBJECTIVES: Cancer-associated fibroblasts (CAFs) are vital constituents of the tumor microenvironment (TME) and play a predominant role in oral squamous cell carcinoma (OSCC) progression. We aimed to investigate the effect and mechanism of exosomal miR-146b-5p derived from CAFs on the malignant biological behavior of OSCC. MATERIALS AND METHODS: Illumina small RNA (sRNA) sequencing was conducted to determine the differential expression patterns of microRNAs (miRNAs) in exosomes derived from CAFs and normal fibroblasts (NFs). Transwell and cell counting kit-8 (CCK-8) assays and xenograft tumor models in nude mice were used to investigate the effect of CAF exosomes and miR-146b-p on the malignant biological behavior of OSCC. Reverse transcription quantitative real-time PCR (qRT-PCR), luciferase reporter, western blotting (WB) and immunohistochemistry assays were employed to investigate the underlying mechanisms involved in CAF exosomes that promote OSCC progression. RESULTS: We demonstrated that CAF-derived exosomes were taken up by OSCC cells and enhanced the proliferation, migration, and invasion ability of OSCC. Compared with NFs, the expression of miR-146b-5p was increased in exosomes and their parent CAFs. Further studies showed that the decreased expression of miR-146b-5p inhibited the proliferation, migration and invasion ability of OSCC cells in vitro and the growth of OSCC cells in vivo. Mechanistically, miR-146b-5p overexpression led to the suppression of HIKP3 by directly targeting the 3'-UTR of HIPK3, as confirmed by luciferase assay. Reciprocally, HIPK3 knockdown partially reversed the inhibitory effect of the miR-146b-5p inhibitor on the proliferation, migration, and invasion ability of OSCC cells and restored their malignant phenotype. CONCLUSIONS: Our results revealed that CAF-derived exosomes contained higher levels of miR-146b-5p than NFs, and miR-146b-5p overexpression in exosomes promoted the malignant phenotype of OSCC by targeting HIPK3. Therefore, inhibiting exosomal miR-146b-5p secretion may be a promising therapeutic modality for OSCC.

Interleukin-33-Enhanced CXCR4 Signaling Circuit Mediated by Carcinoma-Associated Fibroblasts Promotes Invasiveness of Head and Neck Cancer.

Despite recent advances, treatment for head and neck squamous cell carcinoma (HNSCC) has limited efficacy in preventing tumor progression. We confirmed previously that carcinoma-associated fibroblasts (CAF)-induced interleukin-33 (IL-33) contributed to cancer progression. However, the molecular mechanisms underlying the complex communication network of the tumor microenvironment merited further evaluation. To simulate the IL-33-induced autocrine signaling, stable clones of IL-33-overexpressing HNSCC cells were established. Besides well-established IL-33/ST2 and SDF1/CXCR4 (stromal-derived factor 1/C-X-C motif chemokine receptor 4) signaling, the CAF-induced IL-33 upregulated CXCR4 via cancer cell induction of IL-33 self-production. The IL-33-enhanced-CXCR4 regulatory circuit involves SDF1/CXCR4 signaling activation and modulates tumor behavior. An in vivo study confirmed the functional role of IL-33/CXCR4 in tumor initiation and metastasis. The CXCR4 and/or IL-33 blockade reduced HNSCC cell aggressiveness, with attenuated invasions and metastases. Immunohistochemistry confirmed that IL-33 and CXCR4 expression correlated significantly with disease-free survival and IL-33-CXCR4 co-expression predicted a poor outcome. Besides paracrine signaling, the CAF-induced IL-33 reciprocally enhanced the autocrine cancer-cell self-production of IL-33 and the corresponding CXCR4 upregulation, leading to the activation of SDF1/CXCR4 signaling subsequent to cancer progression. Thus, targeting the IL-33-enhanced-CXCR4 regulatory circuit attenuates tumor aggressiveness and provides a potential therapeutic option for improving the prognosis in HNSCC patients.

The multicellular signalling network of ovarian cancer metastases.

BACKGROUND: Transcoelomic spread is the major route of metastasis of ovarian high-grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumour microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. METHODS: Based on transcriptomic analysis of tumour cells, tumour-associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analysing clinical correlations and potentially pro-metastatic functions, including tumour cell migration, pro-inflammatory signal transduction and TAM expansion. RESULTS: The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumour cells, are the major source of most cytokines and growth factors in the omental microenvironment (n = 176 vs. n = 13). Many of these factors target tumour cells, are linked to metastasis and are associated with a short survival. Likewise, tumour stroma cells play a major role in extracellular-matrix-triggered signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumour cell migration and adhesion via WNT4 which is highly expressed by CAF; (ii) induces pro-tumourigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro-inflammatory signaling, at least in part via a HSP70-NF-κB pathway. CONCLUSIONS: The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.