Cancer-associated Fibroblasts Phenotype Research Articles

Abstract 5397: Characteristics of cancer associated fibroblasts isolated from refractory colorectal cancer using RNA sequence

Abstract Primary tumor location of colorectal cancers (CRCs) is emerging as an important prognostic factor owing to distinct biological features. In fact, the patients with right-side CRCs (R-CRCs), in which primary tumors were resected in our department and followed by systemic chemotherapy, have a poorer prognosis in 2-year overall survival and progression-free survival than those with left-side CRCs (L-CRCs). Furthermore, cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. Here, we isolated the CAFs and normal counterparts from 10 CRCs which were performed radical resection, including 5 L-CRCs and 5 R-CRCs, and analyzed transcriptomic profile of CAFs by RNA-sequencing. Gene Set Enrichment Analysis (GSEA) was performed and inflammatory signatures, such as "HALLMARK IL2 STAT5 SIGNALING"(NOM p-value<0.001, FDR q-value=0.146), "HALLMARK IL6 JAK STAT3 SIGNALING"(NOM p-value=0.014, FDR q-value=0.162), "HALLMARK INTERFERON GAMMA RESPONSE"(NOM p-value=0.032, FDR q-value=0.1726), were enriched in the CAFs isolated from R-CRCs (R-CAFs). In addition to sidedness of CRCs, based on gene expression profiles from bulk tumors, CRCs were classified into four consensus molecular subtypes (CMS1-4). Among them, The fibrotic CMS4 subtype portends poor relapse-free survival. We identified 4 CMS4-CRCs, which have the characteristics of stromal activation and the worst prognosis among other CRCs. GSEA showed that the signature of "HALLMARK TNFA_SIGNALING_VIA_NFKB" (NOM p-value=0.023 FDR q-value=0.207), "HALLMARK GLYCOLYSIS" (NOM p-value=0.036 FDR q-value=0.239) were upregulated in the CAFs isolated from CMS4-CRCs. These results indicated that CAFs isolated from refractory CRCs have unique phenotypes and the elucidation of these mechanisms might lead to a promising treatment for refractory CRCs. Citation Format: Hiroaki Kasashima, Yasuhiro Fukui, Ken Yonemitsu, Kisyu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Shigeru Ree, Masakazu Yashiro, Kiyoshi Maeda. Characteristics of cancer associated fibroblasts isolated from refractory colorectal cancer using RNA sequence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5397.

Application of Doxorubicin-loaded PLGA nanoparticles targeting both tumor cells and cancer-associated fibroblasts on 3D human skin equivalents mimicking melanoma and cutaneous squamous cell carcinoma

Nanoparticle (NP) use in cancer therapy is extensively studied in skin cancers. Cancer-associated fibroblasts (CAFs), a major tumor microenvironment (TME) component, promote cancer progression, making dual targeting of cancer cells and CAFs an effective therapy. However, dual NP-based targeting therapy on both tumor cells and CAFs is poorly investigated in skin cancers. Herein, we prepared and characterized doxorubicin-loaded PLGA NPs (DOX@PLGA NPs) and studied their anti-tumor effects on cutaneous melanoma (SKCM)(AN, M14) and cutaneous squamous cell carcinoma (cSCC) (MET1, MET2) cell lines in monolayer, as well as their impact on CAF deactivation. Then, we established 3D full thickness models (FTM) models of SKCM and cSCC using AN or MET2 cells on dermis matrix populated with CAFs respectively, and assessed the NPs' tumor penetration, tumor-killing ability, and CAF phenotype regulation through both topical administration and intradermal injection. The results show that, in monolayer, DOX@PLGA NPs inhibited cancer cell growth and induced apoptosis in a dose- and time-dependent manner, with a weaker effect on CAFs. DOX@PLGA NPs reduced CAF-marker expression and had successful anti-tumor effects in 3D skin cancer FTMs, with decreased tumor-load and invasion. DOX@PLGA NPs also showed great delivery potential in the FTMs and could be used as a platform for future functional study of NPs in skin cancers using human-derived skin equivalents. This study provides promising evidence for the potential of DOX@PLGA NPs in dual targeting therapy for SKCM and cSCC.

Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy.

Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell-targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)-Wnt/β-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer-bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/β-catenin signaling constitutes a promising approach to augment immunotherapy.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions

BackgroundDespite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets.MethodsWe collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis.ResultsThe bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption.ConclusionsThese results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

A phase 1 trial of combined MEK, STAT3 and PD-1 inhibition in metastatic pancreatic ductal adenocarcinoma (PDAC).

TPS713 Background: PDAC is characterized by its innate and acquired resistance to both MAPK pathway inhibition and immune checkpoint (e.g. PD-1/PD-L1) inhibition (ICI) via multiple mechanisms. In preclinical models of PDAC, combined MEK and STAT3 inhibition (MEKi+STAT3i) uncovers stromal plasticity by attenuating cancer-associated fibroblasts (CAF) with IL-6/CXCL1-secretory phenotypes while enriching for Ly6a/CD34-expressing CAF phenotypes with mesenchymal stem cell-like features. This remodeling of CAF heterogeneity is associated with a striking attenuation in and reprogramming of tumor-associated macrophages (TAMs) as well as enhanced trafficking of CD8 T cells, which exhibit a distinct effector and anti-apoptotic transcriptional program. The addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly enhancing the recruitment, degranulating capacity, and functional cytotoxicity of CD8+ T-cells, thereby augmenting antitumor responses and dramatically improving survival in these models. Furthermore, a patient with refractory PDAC treated off-label with this combination achieved a meaningful response. Based on this strong rationale, a phase 1 trial was initiated to test the combination of MEKi+STAT3 and PD1 inhibition in patients with metastatic PDAC. Methods: NCT05440942 is an open-label, prospective, single-institution phase 1 trial testing the safety, preliminary efficacy, and biomarkers of response to the combination of trametinib (MEKi), ruxolitinib (JAK2/STAT3 inhibitor) and retifanlimab (PD-1 inhibitor) in patients with metastatic PDAC. Patients with metastatic PDAC who have had disease progression on at least one line of prior therapy, with good organ function, preserved performance status, and without major intercurrent illness are eligible. Patients must have an accessible lesion for biopsy and must be willing to undergo this research biopsy at baseline and on treatment. Part 1 of the study is a dose-escalation phase with 3 dose levels and a target dose of trametinib 2mg orally daily, ruxolitinib 15mg orally twice daily, and retifanlimab 500mg intravenously every 28 days. The dose escalation is being done using the novel Bayesian keyboard design and 9-15 patients will be treated to get to the potential maximum tolerated dose (MTD). Dose level 1 has been completed without any dose-limiting toxicities seen. Part 2 is an expansion phase which will accrue an additional 20 patients. All patients in part 1 and 2 will have core-needle biopsies pre-treatment and after 4 weeks. Serial blood samples will be collected at baseline and on treatment. Biopsies are being analyzed by multiparameter immune profiling using mass cytometry and bulk RNA sequencing; blood is being analyzed for circulating tumor DNA and immune profiling. These results will be correlated with clinical response to therapy to determine biomarkers of response and resistance. Clinical trial information: NCT05440942 .

Cancer-associated fibroblast phenotypes are associated with patient outcome in non-small cell lung cancer

Despite advances in treatment, lung cancer survival rates remain low. A better understanding of the cellular heterogeneity and interplay of cancer-associated fibroblasts (CAFs) within the tumor microenvironment will support the development of personalized therapies. We report a spatially resolved single-cell imaging mass cytometry (IMC) analysis of CAFs in a non-small cell lung cancer cohort of 1,070 patients. We identify four prognostic patient groups based on 11 CAF phenotypes with distinct spatial distributions and show that CAFs are independent prognostic factors for patient survival. The presence of tumor-like CAFs is strongly correlated with poor prognosis. In contrast, inflammatory CAFs and interferon-response CAFs are associated with inflamed tumor microenvironments and higher patient survival. High density of matrix CAFs is correlated with low immune infiltration and is negatively correlated with patient survival. In summary, our data identify phenotypic and spatial features of CAFs that are associated with patient outcome in NSCLC.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer [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 C025.

Single-cell transcriptome analysis reveals tumoral microenvironment heterogenicity and hypervascularization in human carotid body tumor.

Carotid body tumor (CBT) is a rare neck tumor located at the adventitia of the common carotid artery bifurcation. The prominent pathological features of CBT are high vascularization and abnormal proliferation. However, single-cell transcriptome analysis of the microenvironment composition and molecular complexity in CBT has yet to be performed. In this study, we performed single-cell RNA sequencing (scRNA-seq) analysis on human CBT to define the cells that contribute to hypervascularization and chronic hyperplasia. Unbiased clustering analysis of transcriptional profiles identified 16 distinct cell populations including endothelial cells (ECs), smooth muscle cells (SMCs), neuron cells, macrophage cells, neutrophil cells, and T cells. Within the ECs population, we defined subsets with angiogenic capacity plus clear signs of later endothelial progenitor cells (EPCs) to normal ECs. Two populations of macrophages were detectable in CBT, macrophage1 showed enrichment in hypoxia-inducible factor-1 (HIF-1) and as well as an early EPCs cell-like population expressing CD14 and vascular endothelial growth factor. In addition to HIF-1-related transcriptional protein expression, macrophages1 also display a neovasculogenesis-promoting phenotype. SMCs included threepopulations showing platelet-derived growth factor receptor betaand vimentinexpression, indicative of a cancer-associated fibroblastphenotype. Finally, we identified three types of neuronal cells, including chief cells and sustentacular cells, and elucidated their distinct roles in the pathogenesis of CBT and abnormal proliferation of tumors. Overall, our study provided the first comprehensive characterization of the transcriptional landscape of CBT at scRNA-seq profiles, providing novel insights into the mechanisms underlying its formation.

PDGF-BB accelerates TSCC via fibroblast lactates limiting miR-26a-5p and boosting mitophagy

The tumor microenvironment and cancer-associated fibroblasts (CAFs) play crucial roles in tumor development, and their metabolic coupling remains unclear. Clinical data showed a positive correlation between PDGF-BB, CAFs, and glycolysis in the tumor microenvironment of oral tongue squamous cell carcinoma patients. In vitro, CAFs are derived from hOMF cells treated with PDGF-BB, which induces their formation and promotes aerobic glycolysis. Mitophagy increased the PDGF-BB-induced formation of CAF phenotypes and aerobic glycolysis, while autophagy inhibition blocked PDGF-BB-induced effects. Downregulation of miR-26a-5p was observed in CAFs; upregulation of miR-26a-5p inhibited the expression of mitophagy-related proteins ULKI, Parkin, PINK1, and LC3 and aerobic glycolysis in PDGF-BB-induced CAFs. PDGF-BB-induced CAFs promoted tumor cell proliferation, invasion, metastasis, NF-κB signaling pathway activation, and PDGF-BB secretion. Thus, PDGF-BB is associated with lactate-induced CAF formation and glucose metabolism reprogramming. These findings indicate potential therapeutic targets in oral tongue squamous cell carcinoma.

MiR-200c reprograms fibroblasts to recapitulate the phenotype of CAFs in breast cancer progression.

Mesenchymal-epithelial plasticity driving cancer progression in cancer-associated fibroblasts (CAFs) is undetermined. This work identifies a subgroup of CAFs in human breast cancer exhibiting mesenchymal-to-epithelial transition (MET) or epithelial-like profile with high miR-200c expression. MiR-200c overexpression in fibroblasts is sufficient to drive breast cancer aggressiveness. Oxidative stress in the tumor microenvironment induces miR-200c by DNA demethylation. Proteomics, RNA-seq and functional analyses reveal that miR-200c is a novel positive regulator of NFκB-HIF signaling via COMMD1 downregulation and stimulates pro-tumorigenic inflammation and glycolysis. Reprogramming fibroblasts toward MET via miR-200c reduces stemness and induces a senescent phenotype. This pro-tumorigenic profile in CAFs fosters carcinoma cell resistance to apoptosis, proliferation and immunosuppression, leading to primary tumor growth, metastases, and resistance to immuno-chemotherapy. Conversely, miR-200c inhibition in fibroblasts restrains tumor growth with abated oxidative stress and an anti-tumorigenic immune environment. This work determines the mechanisms by which MET in CAFs via miR-200c transcriptional enrichment with DNA demethylation triggered by oxidative stress promotes cancer progression. CAFs undergoing MET trans-differentiation and senescence coordinate heterotypic signaling that may be targeted as an anti-cancer strategy.

Immunological signatures from irradiated cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are abundant and influential elements of the tumor microenvironment (TME), giving support to tumor development in multiple ways. Among other mechanisms, CAFs are important regulators of immunological processes occurring in tumors. However, CAF-mediated tumor immunomodulation in the context of radiotherapy remains poorly understood. In this study, we explore effects of radiation on CAF-derived immunoregulatory signals to the TME. Primary CAF cultures were established from freshly collected human NSCLC lung tumors. CAFs were exposed to single-high or fractionated radiation regimens (1x18Gy or 3x6Gy), and the expression of different immunoregulatory cell-associated and secreted signaling molecules was analyzed 48h and 6 days after initiation of treatment. Analyses included quantitative measurements of released damage-associated molecular patterns (DAMPs), interferon (IFN) type I responses, expression of immune regulatory receptors, and secretion of soluble cytokines, chemokines, and growth factors. CAFs are able to survive ablative radiation regimens, however they enter into a stage of premature cell senescence. Our data show that CAFs avoid apoptosis and do not contribute by release of DAMPs or IFN-I secretion to radiation-mediated tumor immunoregulation. Furthermore, the secretion of relevant immunoregulatory cytokines and growth factors including TGF-β, IL-6, IL-10, TNFα, IL-1β, VEGF, CXCL12, and CXCL10 remain comparable between non-irradiated and radiation-induced senescent CAFs. Importantly, radiation exposure modifies the cell surface expression of some key immunoregulatory receptors, including upregulation of CD73 and CD276. Our data suggest that CAFs do not participate in the release of danger signals or IFN-I secretion following radiotherapy. The immune phenotype of CAFs and radiation-induced senescent CAFs is similar, however, the observed elevation of some cell surface immunological receptors on irradiated CAFs could contribute to the establishment of an enhanced immunosuppressive TME after radiotherapy.

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma.

Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively. We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples. Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research.

Abstract IA008: Mechanisms and consequences of pancreatic cancer stromal evolution

Abstract The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. We have developed a novel mouse model to track PSC differentiation and function during pancreatic tumor progression in vivo. This model revealed that PSC-derived CAFs in fact give rise to a numerically minor but functionally significant subset of PDAC CAFs, and may represent a viable therapeutic target. We adapted our mouse model to enable targeted ablation of PSCs and derivative CAFs within their host tissue for the first time. Functional studies in this model reveal non-redundant functions for PSC-derived CAFs in shaping the PDAC microenvironment, and highlight the significance of mesenchymal lineage heterogeneity for pancreatic tumorigenesis. Further, we find that tumor genotype with respect to p53 status dictates stromal evolutionary routes with respect to cell of origin, a finding that may extend to additional cancer cell-intrinsic genomic alterations. Together, these findings shed light on mechanisms that shape the tumor microenvironment during pancreatic cancer progression, and may hold relevance in additional solid tumor types. Citation Format: Mara Sherman. Mechanisms and consequences of pancreatic cancer stromal evolution [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA008.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 μM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-β, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Up-Regulation of TCF21 Expression Reverses the Malignant Phenotype of Cancer-Associated Fibroblasts in Esophageal Squamous Cell Carcinoma

Cancer-associated fibroblasts (CAFs), as one major component of tumor microenvironment (TME), are closely associated with tumor initiation and progression. Our previous studies have discovered that CAFs induced the resistance of esophageal squamous cell carcinoma (ESCC) cells to a variety of chemotherapeutic drugs such as cisplatin and paclitaxel. Furthermore, CAFs attenuated ionizing irradiation (IR)-induced cancer cells death by regulating DNA damage response. CAFs themselves are highly resistant to death stimuli due to enhanced antioxidant potential and DNA repair capacity. How to inhibit the malignant phenotype of CAFs is critically important for the radical treatment of ESCC. By RNA-sequencing and DNA methylation analysis, the transcriptome and epigenome of CAFs and matched normal fibroblasts (NFs) have been integratively analyzed. By transfection of TCF21 cDNA plasmid, the expression of TCF21 in CAFs has been up-regulated. Using a cell counting kit and migration and invasion assay, the effect of TCF21 on the growth and migration and invasive ability of CAFs has been detected. Using immunofluorescence and flow cytometry (FCM) analysis and western blotting, the effect of TCF21 on the DNA damage repair and apoptotic death of CAFs following IR has been detected. TCF21 is one of the top ten down-regulated genes in CAFs compared with NFs due to promoter methylation. Up-regulation of TCF21 expression inhibited the growth rate and migration and invasive ability of CAFs. The expression of α-SMA, as an indicator of CAFs activation, was down-regulated in CAFs which were transfected with TCF21 cDNA. Furthermore, when TCF21 cDNA was transfected into CAFs, IR-induced DNA damage was increased while DNA repair was inhibited in CAFs, suggesting that TCF21 was involved in DNA damage response of CAFs following IR. FCM analysis showed that up-regulation of TCF21 expression promoted IR-induced apoptotic death of CAFs. TCF21 is a determinant of the malignant phenotype of CAFs in ESCC. Up-regulation of TCF21 expression is a promising approach of inhibiting the growth, migration and invasion, activation and radioresistance of CAFs in ESCC.

Low-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models

Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.

Cutaneous squamous cell carcinoma-derived extracellular vesicles exert an oncogenic role by activating cancer-associated fibroblasts

Cutaneous squamous cell carcinoma (cSCC) is a fast-increasing cancer with metastatic potential. Extracellular vesicles (EVs) are small membrane-bound vesicles that play important roles in intercellular communication, particularly in the tumor microenvironment (TME). Here we report that cSCC cells secrete an increased number of EVs relative to normal human epidermal keratinocytes (NHEKs) and that interfering with the capacity of cSCC to secrete EVs inhibits tumor growth in vivo in a xenograft model of human cSCC. Transcriptome analysis of tumor xenografts by RNA-sequencing enabling the simultaneous quantification of both the human and the mouse transcripts revealed that impaired EV-production of cSCC cells prominently altered the phenotype of stromal cells, in particular genes related to extracellular matrix (ECM)-formation and epithelial-mesenchymal transition (EMT). In line with these results, co-culturing of human dermal fibroblasts (HDFs) with cSCC cells, but not with normal keratinocytes in vitro resulted in acquisition of cancer-associated fibroblast (CAF) phenotype. Interestingly, EVs derived from metastatic cSCC cells, but not primary cSCCs or NHEKs, were efficient in converting HDFs to CAFs. Multiplex bead-based flow cytometry assay and mass-spectrometry (MS)-based proteomic analyses revealed the heterogenous cargo of cSCC-derived EVs and that especially EVs derived from metastatic cSCCs carry proteins associated with EV-biogenesis, EMT, and cell migration. Mechanistically, EVs from metastatic cSCC cells result in the activation of TGFβ signaling in HDFs. Altogether, our study suggests that cSCC-derived EVs mediate cancer-stroma communication, in particular the conversion of fibroblasts to CAFs, which eventually contribute to cSCC progression.

An HGF-dependent positive feedback loop between bladder cancer cells and fibroblasts mediates lymphangiogenesis and lymphatic metastasis.

Cancer-associated fibroblasts (CAFs) play a vital role in facilitating tumor progression through extensive reciprocal interplay with cancer cells. Tumor-derived extracellular vesicles (EVs) are the critical mediators involved in the crosstalk between cancer cells and stromal cells, contributing to the metastasis of cancers. Yet, the biological mechanisms of tumor-derived EVs in triggering CAFs phenotype to stimulate the lymph node (LN) metastasis of bladder cancer (BCa) are largely unknown. Here, we aimed to explore the effects and molecular mechanisms of tumor-derived EV-mediated CAFs phenotype in regulating BCa LN metastasis. The high-throughput sequencing was utilized to identify the crucial long non-coding RNA (lncRNA) associated with CAF enrichment in BCa. The functional role of the transition of fibroblasts to CAFs induced by LINC00665-mediated EVs was investigated through the in vitro and in vivo assays. Chromatin isolation by RNA purification assays, fluorescence resonance energy transfer assays, cytokine profiling and patient-derived xenograft (PDX) model were performed to explore the underlying mechanism of LINC00665 in the LN metastasis of BCa. We found that CAFs are widely enriched in the tumor microenvironment of BCa, which correlated with BCa lymphangiogenesis and LN metastasis. We then identified a CAF-associated long non-coding RNA, LINC00665, which acted as a crucial mediator of CAF infiltration in BCa. Clinically, LINC00665 was associated with LN metastasis and poor prognosis in patients with BCa. Mechanistically, LINC00665 transcriptionally upregulated RAB27B expression and induced H3K4me3 modification on the promoter of RAB27B through the recruitment of hnRNPL. Moreover, RAB27B-induced EVs secretion endowed fibroblasts with the CAF phenotype, which reciprocally induced LINC00665 overexpression to form a RAB27B-HGF-c-Myc positive feedback loop, enhancing the lymphangiogenesis and LN metastasis of BCa. Importantly, we demonstrated that blocking EV-transmitted LINC00665 or HGF broke this loop and impaired BCa lymphangiogenesis in a PDX model. Our study uncovers a precise mechanism that LINC00665 sustains BCa LN metastasis by inducing a RAB27B-HGF-c-Myc positive feedback loop between BCa cells and fibroblasts, suggesting that LINC00665 could be a promising therapeutic target for patients with LN metastatic BCa.

Hepatocellular carcinoma cells remodel the pro-metastatic tumour microenvironment through recruitment and activation of fibroblasts via paracrine Egfl7 signaling

BackgroundThe tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear.MethodsPrimary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model.ResultsWe showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανβ3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis.ConclusionOur data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανβ3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients.3o7rdHzTehVwN25qa8k-ATVideo

Cancer-associated fibroblast classification in single-cell and spatial proteomics data

Cancer-associated fibroblasts (CAFs) are a diverse cell population within the tumour microenvironment, where they have critical effects on tumour evolution and patient prognosis. To define CAF phenotypes, we analyse a single-cell RNA sequencing (scRNA-seq) dataset of over 16,000 stromal cells from tumours of 14 breast cancer patients, based on which we define and functionally annotate nine CAF phenotypes and one class of pericytes. We validate this classification system in four additional cancer types and use highly multiplexed imaging mass cytometry on matched breast cancer samples to confirm our defined CAF phenotypes at the protein level and to analyse their spatial distribution within tumours. This general CAF classification scheme will allow comparison of CAF phenotypes across studies, facilitate analysis of their functional roles, and potentially guide development of new treatment strategies in the future.