Expression In Cancer-associated Fibroblasts Research Articles

CPT1C-positive cancer-associated fibroblast facilitates immunosuppression through promoting IL-6-induced M2-like phenotype of macrophage

ABSTRACT Cancer-associated fibroblasts (CAFs) exhibit remarkable phenotypic heterogeneity, with specific subsets implicated in immunosuppression in various malignancies. However, whether and how they attenuate anti-tumor immunity in gastric cancer (GC) remains elusive. CPT1C, a unique isoform of carnitine palmitoyltransferase pivotal in regulating fatty acid oxidation, is briefly indicated as a protumoral metabolic mediator in the tumor microenvironment (TME) of GC. In the present study, we initially identified specific subsets of fibroblasts exclusively overexpressing CPT1C, hereby termed them as CPT1C+CAFs. Subsequent findings indicated that CPT1C+CAFs fostered a stroma-enriched and immunosuppressive TME as they correlated with extracellular matrix-related molecular features and enrichment of both immunosuppressive subsets, especially M2-like macrophages, and multiple immune-related pathways. Next, we identified that CPT1C+CAFs promoted the M2-like phenotype of macrophage in vitro. Bioinformatic analyses unveiled the robust IL-6 signaling between CPT1C+CAFs and M2-like phenotype of macrophage and identified CPT1C+CAFs as the primary source of IL-6. Meanwhile, suppressing CPT1C expression in CAFs significantly decreased IL-6 secretion in vitro. Lastly, we demonstrated the association of CPT1C+CAFs with therapeutic resistance. Notably, GC patients with high CPT1C+CAFs infiltration responded poorly to immunotherapy in clinical cohort. Collectively, our data not only present the novel identification of CPT1C+CAFs as immunosuppressive subsets in TME of GC, but also reveal the underlying mechanism that CPT1C+CAFs impair tumor immunity by secreting IL-6 to induce the immunosuppressive M2-like phenotype of macrophage in GC.

Stable and oscillatory hypoxia differentially regulate invasibility of breast cancer associated fibroblasts

As local regions in the tumor outstrip their oxygen supply, hypoxia can develop, affecting not only the cancer cells, but also other cells in the microenvironment, including cancer associated fibroblasts (CAFs). Hypoxia is also not necessarily stable over time, and can fluctuate or oscillate. Hypoxia Inducible Factor-1 is the master regulator of cellular response to hypoxia, and can also exhibit oscillations in its activity. To understand how stable, and fluctuating hypoxia influence breast CAFs, we measured changes in gene expression in CAFs in normoxia, hypoxia, and oscillatory hypoxia, as well as measured change in their capacity to resist, or assist breast cancer invasion. We show that hypoxia has a profound effect on breast CAFs causing activation of key pathways associated with fibroblast activation, but reduce myofibroblast activation and traction force generation. We also found that oscillatory hypoxia, while expectedly resulted in a “sub-hypoxic” response in gene expression, it resulted in specific activation of pathways associated with actin polymerization and actomyosin maturation. Using traction force microscopy, and a nanopatterned stromal invasion assay, we show that oscillatory hypoxia increases contractile force generation vs stable hypoxia, and increases heterogeneity in force generation response, while also additively enhancing invasibility of CAFs to MDA-MB-231 invasion. Our data show that stable and unstable hypoxia can regulate many mechnobiological characteristics of CAFs, and can contribute to transformation of CAFs to assist cancer dissemination and onset of metastasis.

Identification of BGN positive fibroblasts as a driving factor for colorectal cancer and development of its related prognostic model combined with machine learning

BackgroundNumerous studies have indicated that cancer-associated fibroblasts (CAFs) play a crucial role in the progression of colorectal cancer (CRC). However, there are still many unknowns regarding the exact role of CAF subtypes in CRC.MethodsThe data for this study were obtained from bulk, single-cell, and spatial transcriptomic sequencing data. Bioinformatics analysis, in vitro experiments, and machine learning methods were employed to investigate the functional characteristics of CAF subtypes and construct prognostic models.ResultsOur study demonstrates that Biglycan (BGN) positive cancer-associated fibroblasts (BGN + Fib) serve as a driver in colorectal cancer (CRC). The proportion of BGN + Fib increases gradually with the progression of CRC, and high infiltration of BGN + Fib is associated with poor prognosis in terms of overall survival (OS) and recurrence-free survival (RFS) in CRC. Downregulation of BGN expression in cancer-associated fibroblasts (CAFs) significantly reduces migration and proliferation of CRC cells. Among 101 combinations of 10 machine learning algorithms, the StepCox[both] + plsRcox combination was utilized to develop a BGN + Fib derived risk signature (BGNFRS). BGNFRS was identified as an independent adverse prognostic factor for CRC OS and RFS, outperforming 92 previously published risk signatures. A Nomogram model constructed based on BGNFRS and clinical-pathological features proved to be a valuable tool for predicting CRC prognosis.ConclusionIn summary, our study identified BGN + Fib as drivers of CRC, and the derived BGNFRS was effective in predicting the OS and RFS of CRC patients.

Metformin suppresses hypoxia-inducible factor-1α expression in cancer-associated fibroblasts to block tumor-stromal cross-talk in breast cancer

To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer. Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-β1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs. Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformintreated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OGinduced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05). Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Beyond Small Molecules: Antibodies and Peptides for Fibroblast Activation Protein Targeting Radiopharmaceuticals.

Fibroblast activation protein (FAP) is a serine protease characterized by its high expression in cancer-associated fibroblasts (CAFs) and near absence in adult normal tissues and benign lesions. This unique expression pattern positions FAP as a prospective biomarker for targeted tumor radiodiagnosis and therapy. The advent of FAP-based radiotheranostics is anticipated to revolutionize cancer management. Among various types of FAP ligands, peptides and antibodies have shown advantages over small molecules, exemplifying prolonged tumor retention in human volunteers. Within its scope, this review summarizes the recent research progress of the FAP radiopharmaceuticals based on antibodies and peptides in tumor imaging and therapy. Additionally, it incorporates insights from recent studies, providing valuable perspectives on the clinical utility of FAP-targeted radiopharmaceuticals.

Tumor-to-stroma cd8+ t cells ratio combined with cancer-associated fibroblasts: an innovative approach to predicting lymph node metastases of cervical cancer

PurposePrecise identification of lymph node metastases is vital for the management of cervical cancer. However, the existing diagnostic methods for lymph node metastases have certain drawbacks. In this study, we aim to explore the expression of cancer-associated fibroblasts (CAFs) and tumor-to-stroma CD8+ T cells ratio (CD8+ T cells T:S ratio) and its association with lymph node metastases of cervical cancer.MethodsHundred and ten cervical cancer tissues and 39 biopsy tissues from patients were investigated immunocytochemically for the expression of CAFs and CD8+ T cells. The statistical correlation analysis was carried out using the SPSS system.ResultsA strong and statistically significant negative correlation (r= − 0.690; P < 0.001) was observed between CAF density and CD8+ T cells T:S ratio. Not only were CAFs density and CD8+ T cells T:S ratio correlated with lymph node metastases respectively (P < 0.001), but the combination of them also significantly correlated with lymph node metastases (P < 0.001). Then, we constructed the combined diagnosis model (Logit (P) = − 4.446 + 0.300 × CAFs + 0.752 × CD8+ T cells T:S Ratio) of cervical cancer lymph node metastases. ROC curves analysis showed that the ROC curves areas for CAFs, CD8+ T cells T:S ratio, and a combination of both are 0.879, 0.747, and 0.951. Then, the prediction model was verified by biopsy specimens and consistent results were obtained.ConclusionsThe combination of CAF density and CD8+ T cells T:S ratio has a significant predictive value for lymph node metastases in patients with cervical cancer.

Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC.

Cancer-associated fibroblasts (CAFs) play key roles in the tumor microenvironment. IgA contributes to inflammation and dismantling antitumor immunity in the human liver. In this study, we aimed to elucidate the effects of the IgA complex on CAFs in Pil Soo Sung the tumor microenvironment of HCC. CAF dynamics in HCC tumor microenvironment were analyzed through single-cell RNA sequencing of HCC samples. CAFs isolated from 50 HCC samples were treated with mock or serum-derived IgA dimers in vitro. Progression-free survival of patients with advanced HCC treated with atezolizumab and bevacizumab was significantly longer in those with low serum IgA levels ( p <0.05). Single-cell analysis showed that subcluster proportions in the CAF-fibroblast activation protein-α matrix were significantly increased in patients with high serum IgA levels. Flow cytometry revealed a significant increase in the mean fluorescence intensity of fibroblast activation protein in the CD68 + cells from patients with high serum IgA levels ( p <0.001). We confirmed CD71 (IgA receptor) expression in CAFs, and IgA-treated CAFs exhibited higher programmed death-ligand 1 expression levels than those in mock-treated CAFs ( p <0.05). Coculture with CAFs attenuated the cytotoxic function of activated CD8 + T cells. Interestingly, activated CD8 + T cells cocultured with IgA-treated CAFs exhibited increased programmed death-1 expression levels than those cocultured with mock-treated CAFs ( p <0.05). Intrahepatic IgA induced polarization of HCC-CAFs into more malignant matrix phenotypes and attenuates cytotoxic T-cell function. Our study highlighted their potential roles in tumor progression and immune suppression.

ANKRD1 is a mesenchymal-specific driver of cancer-associated fibroblast activation bridging androgen receptor loss to AP-1 activation

There are significant commonalities among several pathologies involving fibroblasts, ranging from auto-immune diseases to fibrosis and cancer. Early steps in cancer development and progression are closely linked to fibroblast senescence and transformation into tumor-promoting cancer-associated fibroblasts (CAFs), suppressed by the androgen receptor (AR). Here, we identify ANKRD1 as a mesenchymal-specific transcriptional coregulator under direct AR negative control in human dermal fibroblasts (HDFs) and a key driver of CAF conversion, independent of cellular senescence. ANKRD1 expression in CAFs is associated with poor survival in HNSCC, lung, and cervical SCC patients, and controls a specific gene expression program of myofibroblast CAFs (my-CAFs). ANKRD1 binds to the regulatory region of my-CAF effector genes in concert with AP-1 transcription factors, and promotes c-JUN and FOS association. Targeting ANKRD1 disrupts AP-1 complex formation, reverses CAF activation, and blocks the pro-tumorigenic properties of CAFs in an orthotopic skin cancer model. ANKRD1 thus represents a target for fibroblast-directed therapy in cancer and potentially beyond.

YAP1 controls the N-cadherin-mediated tumor-stroma interaction in melanoma progression

The hallmark of epithelial-to-mesenchymal transition (EMT) is the switch from epithelial cadherin (E-cadherin) to neural cadherin (N-cadherin), allowing melanoma cells to form a homotypic N-cadherin-mediated adhesion with stromal fibroblasts. However, how cadherin switching is initiated, maintained, and regulated in melanoma remains elusive. Here, we report a novel mechanism underlying cadherin switching in melanoma cells that is regulated by stromal Yes-associated protein 1 (YAP1) signaling. The progression of a BRAF-mutant mouse melanoma was suppressed in vivo upon YAP1 ablation in cancer-associated fibroblasts (CAFs). On the contrary, overexpressing YAP1 in CAFs accelerated melanoma development. By RNA-Seq, N-cadherin was identified as a major downstream effector of YAP1 signaling in CAFs. YAP1 silencing reduced N-cadherin expression in CAFs, leading to the downregulation of N-cadherin in neighboring melanoma cells. N-cadherin ablation inhibited the PI3K-AKT signaling pathway in melanoma cells and melanoma cell proliferation. The findings suggest that YAP1 depletion in CAFs induces the downregulation of p-AKT signaling in melanoma cells through the N-cadherin-mediated interaction between melanoma cells and CAFs. The data underscore an important role of CAFs in regulating N-cadherin-mediated adhesion and signaling in melanoma and highlight that disentangling cadherin-mediated cell-cell interactions can potentially disrupt tumor-stroma interactions and reverse the tumor cell invasive phenotype.

Cancer-associated fibroblasts contributed to hepatocellular carcinoma recurrence and metastasis via CD36-mediated fatty-acid metabolic reprogramming

Cancer-associated fibroblasts (CAFs) are the main components in the tumor microenvironment. Tumors activate fibroblasts from quiescent state into activated state by secreting cytokines, and activated CAFs may in turn promote tumor progression and metastasis. Therefore, studies targeting CAFs could enrich the therapeutic options for tumor treatment. In this study, we demonstrate that the content of lipid droplets and the expression of autophagosomes were higher in CAFs than in peri-tumor fibroblasts (PTFs), which was inhibited by 5-(tetradecyloxy)-2-furoic acid(TOFA). The expression of CD36 in CAFs was higher than that in PTFs at both mRNA and protein levels. Inhibition of CD36 activity using either the CD36 inhibitor SSO or siRNA had a significant negative impact on the proliferation and migration abilities of CAFs, which was associated with reduced levels of relevant activated genes (α-SMA, FAP, Vimentin) and cytokines (IL-6, TGF-β and VEGF-α). SSO also inhibited HCC growth and tumorigenesis in nude mice orthotopically implanted with CAFs and HCC cells. Our data further show that CD36+CAFs affected the expression of PD-1 in CTLs leading to CTL exhaustion, and that patients with high CD36 expression in CAFs were correlated with shorter overall survival (OS). Together, our data demonstrate that CAFs were active in lipid metabolism with increased lipid content and lipophagy activity. CD36 may play a key role in the regulation of the biological behaviors of CAFs, which may influence the proliferation and migration of tumor cells by reprograming the lipid metabolism in tumor cells. Thus, CD36 could be an effective therapeutic target for the treatment of HCC.

AKT2S128/CCTαS315/319/323-positive cancer-associated fibroblasts (CAFs) mediate focal adhesion kinase (FAK) inhibitors resistance via secreting phosphatidylcholines (PCs)

Abnormal metabolism is regarded as an oncogenic hallmark related to tumor progression and therapeutic resistance. Present study employed multi-omics, including phosphoproteomics, untargeted metabolomics and lipidomics, to demonstrate that the pAKT2 Ser128 and pCCTα Ser315/319/323-positive cancer-associated fibroblasts (CAFs) substantially release phosphatidylcholines (PCs), contributing to the resistance of focal adhesion kinase (FAK) inhibitors in esophageal squamous cell carcinoma (ESCC) treatment. Additionally, we observed extremely low levels of FAK Tyr397 expression in CAFs, potentially offering no available target for FAK inhibitors playing their anti-growth role in CAFs. Consequently, FAK inhibitor increased the intracellular concentration of Ca2+ in CAFs, promoting the formation of AKT2/CCTα complex, leading to phosphorylation of CCTα Ser315/319/323 sites and eventually enhancing stromal PC production. This activation could stimulate the intratumoral Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway, triggering resistance to FAK inhibition. Analysis of clinical samples demonstrated that stromal pAKT2 Ser128 and pCCTα Ser315/319/323 are related to the tumor malignancy and reduced patient survival. Pseudo-targeted lipidomics and further validation cohort quantitatively showed that plasma PCs enable to distinguish the malignant extent of ESCC patients. In conclusion, inhibition of stroma-derived PCs and related pathway could be possible therapeutic strategies for tumor therapy.

Abstract C018: Spatial profiling of tumor associated cells expressing endoplasmic reticulum stress proteins predicts poor outcomes in pancreatic cancer patients

Abstract The crosstalk between pancreatic cancer (PC) cells and cells of the tumor microenvironment (TME), such as immune cells and cancer associated fibroblasts (CAFs), has important implications for tumor progression. It is evident that the expression of endoplasmic reticulum (ER) stress proteins (e.g., BiP or CHOP) in tumor cells has survival implications for patients. For example, high BiP expression in tumor cells is correlated with poor survival in PC patients. However, the effect of these proteins in TME cells is currently unknown. We have employed quantitative multiplex immunofluorescence on a tumor microarray cohort of 259 treatment naive pancreatic cancer patient samples and 41 normal pancreas samples. We investigated the impact of expression of ER stress proteins BiP or CHOP in CAFS (α-SMA+), immune cells (CD45+), endothelial cells (CD31+) and tumor cells (PanCK+). HALO AI (Indica Labs) was used to conduct cell/tissue classifications and spatial proximity analysis. Notably, the proportion of CAFs and endothelial cells expressing BiP or CHOP was significantly (p&amp;lt;0.01-0.0001) increased in tumor sections compared to non-tumor sections. When considering the tumor sections, increased BiP expression in CAFs and endothelial cells was found to be correlated with vascular invasion (p=0.0388 and p=0.0318 respectively). Interestingly, there was also a positive correlation between the percent of total CAFs and the percent of BiP positive cells (p=0.0037, r=0.293) or CHOP positive cells (p=0.0001, r=0.235), suggesting that ER stress could be associated with activation of CAFs. Spatial analysis was next utilized to explore the distribution of cells expressing BiP or CHOP within the TME. Remarkably, when examining the average distance in a 500 μm radius, our findings showed that immune cells expressing BiP or CHOP were located significantly (p&amp;lt;0.0001) closer to tumor cells compared to non-expressing immune cells. Interestingly, increased distance of CHOP expressing immune cells to tumor cells was negatively correlated (p=0.037, r=-0.137) with tumor size. We next examined these cells within a 20 μm radius of tumor cells, as it represents a physiologically feasible distance for direct cell-cell contact. Increased numbers of CHOP CD45 cells within the 20 μm radius was correlated with poorer overall survival (p=0.0062) and event free survival (p=0.046). Similarly, in CAFS, BiP expressing cells were found on average closer to tumor cells (p&amp;lt;0.0001), although this was not associated with patient survival. The observed correlation between elevated ER stress in TME cells with adverse patient outcomes indicates the importance of BiP or CHOP as potential therapeutic targets. Furthermore, the spatial analysis results provide insights into the organization of TME cells, shedding light on the significance of ER stress in the TME. These findings contribute to our understanding of the complex interplay between ER stress, tumor biology, and patient outcomes in PC. Citation Format: Georgia M. Porter, Murray Norris, Minoti Apte, Angelica Merlot. Spatial profiling of tumor associated cells expressing endoplasmic reticulum stress proteins predicts poor outcomes in pancreatic cancer patients [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 C018.

Prognostic significance of tumor budding in patients with pancreatic invasive ductal carcinoma who received neoadjuvant therapy

Neoadjuvant therapy is commonly used for invasive pancreatic ductal carcinoma (PDAC). Tumor budding and high podoplanin expression in cancer-associated fibroblasts (CAFs) are prognostic factors in patients with various carcinomas including PDAC who have not received neoadjuvant therapy. In this study, we investigated whether tumor budding and podoplanin-positive CAFs are associated with outcomes in Japanese PDAC patients with neoadjuvant therapy. Histopathological findings of surgically resected PDACs with neoadjuvant therapy from 2005 to 2018 were reviewed (n = 97). With reference to International Tumor Budding Consensus Conference recommendations, tumors were evaluated for budding at 20 × magnification (/0.785 mm2) and at 40 × magnification (/0.237 mm2; mean number of fields: 3) for podoplanin expression in CAFs (%). Overall survival, disease-free survival, and disease-specific survival (DSS) were analyzed using the log-rank test and Cox proportional hazards model. After adjusting for T category, N category, resection margin, and adjuvant therapy, multivariate analyses demonstrated that tumor budding at 40 × magnification was an independent prognostic factor for worse DSS (hazard ratio: 2.41, p = 0.022). Tumor budding at 20 × magnification and podoplanin-positive CAFs tended to be associated with worse DSS; however, these findings were not statistically significant. Our findings indicate that tumor budding is an independent prognostic factor in PDAC patients with neoadjuvant therapy.

Cancer-associated fibroblasts-induced remodeling of tumor immune microenvironment via Jagged1 in glioma

Tumor immunosuppression are prominent characteristics of brain glioma. Current standard modality including surgical resection and chemoradiotherapy do not significantly improve clinical outcomes. Cancer-associated fibroblasts (CAFs) that regard as important stromal cells in tumor microenvironment have been confirmed to play crucial roles in tumor development. However, the effects of CAFs on tumor immunosuppression in glioma are not well expounded. In this study, we report that CAFs contributes to the formation of glioma immunosuppressive microenvironment. Specifically, we found that glioma-derived Jagged1 enhanced the proliferation and PD-L1 expression of CAFs in vitro. Importantly, we discovered that Notch1, c-Myc and PD-L1 expression were significantly increased in high Jagged1-expressing gliomas, moreover, we further confirmed that Notch1 and PD-L1 expression located on the CAFs in glioma tissues. We also found that glioma-derived Jagged1 promotes the increase of tumor-infiltrating macrophages, M2 macrophages and Foxp3 Treg cells, as well as no significance of M1 macrophages and CD8+ T cells, indicating potential immunosuppression. This study opens up novel therapeutic strategies reversing CAF immunosuppression for gliomas.

A prognostic model based on Scissor+ cancer associated fibroblasts identified from bulk and single cell RNA sequencing data in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one of the most lethal diseases in the world, which often recur after multimodality treatment approaches, leading to a poor prognosis. Fibroblasts, a heterogeneous component of the tumor microenvironment, can modulate numerous aspects of tumor biology and have been increasingly acknowledged in dictating the clinical outcome of patients with HNSCC. However, the subpopulation of fibroblasts that are related to the prognosis of HNSCC has not yet been fully explored. To do so, we combined a single-cell RNA sequencing (scRNA-seq) dataset and bulk RNA-sequencing dataset with clinical information, identifying the fibroblast population that are related to poor prognosis of HNSCC. We found these specific population of fibroblasts are less differentiated. In addition, to identify the prognostic signatures of HNSCC, bioinformatics analysis included least absolute shrinkage and selection operator (LASSO) analyses and univariate cox and were performed. We selected 12 prognosis-related genes for constructing a risk model using The Cancer Genome Atlas (TCGA). The AUC values and calibration plots of this model indicated good prognostic prediction efficacy. This model also was validated in two Gene Expression Omnibus (GEO) datasets. In conclusion, we constructed an optimal model that was derived from single cell RNA-seq and bulk RNA-seq to predict the survival probability of HNSCC patients. Among this model, AKR1C3 higher expression in cancer associated fibroblasts (CAFs) of HNSCC has been confirmed by preliminary experiments.

Statins abrogate gemcitabine-induced PD-L1 expression in pancreatic cancer-associated fibroblasts and cancer cells with improved therapeutic outcome.

Acombination of chemotherapy with immunotherapy has been proposed to have better clinical outcomes in Pancreatic Ductal Adenocarcinoma (PDAC). On the other hand, chemotherapeutics is known to have certain unwanted effects on the tumor microenvironment that may mask the expected beneficial effects of immunotherapy. Here, we have investigated the effect of gemcitabine (GEM), on two immune checkpoint proteins (PD-L1 and PD-L2) expression in cancer associated fibroblasts (CAFs) and pancreatic cancer cells (PCCs). Findings of in vitro studies conducted by using in-culture activated mouse pancreatic stellate cells (mPSCs) and human PDAC patients derived CAFs demonstrated that GEM significantly induces PD-L1 and PD-L2 expression in these cells. Moreover, GEM induced phosphorylation of STAT1 and production of multiple known PD-L1-inducing secretory proteins including IFN-γ in CAFs. Upregulation of PD-L1 in PSCs/CAFs upon GEM treatment caused T cell inactivation and apoptosis in vitro. Importantly, Statins suppressed GEM-induced PD-L1 expression both in CAFs and PCCs while abrogating the inactivation of T-cells caused by GEM-treated PSCs/CAFs. Finally, in an immunocompetent syngeneic orthotopic mouse pancreatic tumor model, simvastatin and GEM combination therapy significantly reduced intra-tumor PD-L1 expression and noticeably reduced the overall tumor burden and metastasis incidence. Together, the findings of this study have provided experimental evidence that illustrates potential unwanted side effects of GEM that could hamper the effectiveness of this drug as mono and/or combination therapy. At the same time the findings also suggest use of statins along with GEM will help in overcoming these shortcomings and warrant further clinical investigation.

A novel STAT3/ NFκB p50 axis regulates stromal-KDM2A to promote M2 macrophage-mediated chemoresistance in breast cancer

BackgroundLysine Demethylase 2A (KDM2A) plays a crucial role in cancer cell growth, differentiation, metastasis, and the maintenance of cancer stemness. Our previous study found that cancer-secreted IL-6 can upregulate the expression of KDM2A to promote further the transition of cells into cancer-associated fibroblasts (CAFs). However, the molecular mechanism by which breast cancer-secreted IL-6 regulates the expression of KDM2A remains unclear. Therefore, this study aimed to elucidate the underlying molecular mechanism of IL-6 in regulating KDM2A expression in CAFs and KDM2A-mediated paclitaxel resistance in breast cancer.MethodsThe ectopic vector expression and biochemical inhibitor were used to analyze the KDM2A expression regulated by HS-578 T conditioned medium or IL-6 in mammary fibroblasts. Immunoprecipitation and chromatin immunoprecipitation assays were conducted to examine the interaction between STAT3 and NFκB p50. M2 macrophage polarization was assessed by analyzing M2 macrophage-specific markers using flow cytometry and RT-PCR. ESTIMATE algorithm was used to analyze the tumor microenvironment-dominant breast cancer samples from the TCGA database. The correlation between stromal KDM2A and CD163 + M2 macrophages was analyzed using the Pearson correlation coefficient. Cell viability was determined using trypan blue exclusion assay.ResultsIL-6 regulates gene expression via activation and dimerization of STAT3 or collaboration of STAT3 and NFκB. However, STAT3, a downstream transcription factor of the IL-6 signaling pathway, was directly complexed with NFκB p50, not NFκB p65, to upregulate the expression of KDM2A in CAFs. Enrichment analysis of immune cells/stromal cells using TCGA-breast cancer RNA-seq data unveiled a positive correlation between stromal KDM2A and the abundance of M2 macrophages. CXCR2-associated chemokines secreted by KDM2A-expressing CAFs stimulated M2 macrophage polarization, which in turn secreted CCL2 to increase paclitaxel resistance in breast cancer cells by activating CCR2 signaling.ConclusionThis study revealed the non-canonical molecular mechanism of IL-6 secreted by breast cancer upregulated KDM2A expression in CAFs via a novel STAT3/NFκB p50 axis, which STAT3 complexed with NFκB p50 in NFκB p50 binding motif of KDM2A promoter. KDM2A-expressing CAFs dominantly secreted the CXCR2-associated chemokines to promote M2 macrophage polarization and enhance paclitaxel resistance in breast cancer. These findings underscore the therapeutic potential of targeting the CXCR2 or CCR2 pathway as a novel strategy for paclitaxel-resistant breast cancer.

Anxiolytics cause anxiety in pancreatic cancer

Benzodiazepines (BZDs) are commonly prescribed for pancreatic cancer patients. To investigate the correlation between BZDs and survival outcomes a recent study by Cornwell et al. found that lorazepam (LOR) correlates with poor survival. The mechanistic study shows that LOR increases interleukin 6 (IL6) expression in cancer-associated fibroblasts via GPR68.

Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

CD105 expression in cancer-associated fibroblasts: a biomarker for bone metastasis in early invasive ductal breast cancer patients.

Introduction: Bone metastasis is one of the causes that mainly decrease survival in patients with advanced breast cancer. Therefore, it is essential to find prognostic markers for the occurrence of this type of metastasis during the early stage of the disease. Currently, cancer-associated fibroblasts, which represent 80% of the fibroblasts present in the tumor microenvironment, are an interesting target for studying new biomarkers and developing alternative therapies. This study evaluated the prognostic significance of the CD105 expression in cancer-associated fibroblasts in early breast cancer patients. Methods: Immunohistochemistry was used to assess CD105 expression in invasive ductal breast carcinomas (n = 342), analyzing its association with clinical and pathological characteristics. Results: High CD105 expression in cancer-associated fibroblasts was associated with an increased risk of metastatic occurrence (p = 0.0003), particularly bone metastasis (p = 0.0005). Furthermore, high CD105 expression was associated with shorter metastasis-free survival, bone metastasis-free survival, and overall survival (p = 0.0002, 0.0006, and 0.0002, respectively). CD105 expression also constituted an independent prognostic factor for metastasis-free survival, bone metastasis-free survival, and overall survival (p = 0.0003, 0.0006, and 0.0001, respectively). Discussion: The high CD105 expression in cancer-associated fibroblasts is an independent prognostic marker for bone metastasis in early breast cancer patients. Therefore, the evaluation of CD105(+) CAFs could be crucial to stratify BCPs based on their individual risk profile for the development of BM, enhancing treatment strategies and outcomes.