Proliferation Of Cancer-associated Fibroblasts Research Articles

Research on curcumin mediating immunotherapy of colorectal cancer by regulating cancer associated fibroblasts.

The objective was to investigate curcumin's (Cur) function and associated molecular mechanisms in regulating tumor immunity in colon cancer. Primary cancer-associated fibroblasts (CAFs) from mouse CT26 colon cancer tumors were isolated. Validation of primary CAFs using immunofluorescence assay was done. Cell Counting Kit-8 experiments, real-time quantitative PCR (qPCR), and enzyme linked immunosorbent assay experiments were conducted to investigate how curcumin affected the growth and cytokine secretion functions of CAFs. The effect of curcumin on regulating PD-L1 expression on CT26 cells through CAFs in vitro was explored through coculture of CAFs and tumor cells, qPCR, and western blot experiments. A mouse colon cancer cell model was established in Balb/c nude mice to explore the effect of curcumin on colon tumor cells. Changes in the tumor microenvironment were detected by flow cytometry to explore the synergistic effect of curcumin combined with anti-PD-1 monoclonal antibody in the treatment of mouse colon cancer. In vitro, curcumin prevented the growth and TGF-β secretion of CT26 cells. At the same time, curcumin inhibited the secretion of TGF-β by CAFs, thereby downregulating the PD-L1 expression of CT26 cells. In vivo, curcumin combined with anti-PD-1 antibodies can further enhance the inhibitory effect of PD-1 antibodies on tumors and increase the number of tumor-suppressing immune cells in the tumor microenvironment, such as M1 macrophages and CD8 T cells, thus inhibiting tumors. Immune M2 macrophages, regulatory T cells, and other cells were reduced. In conclusion, curcumin reduces the expression of PD-L1 in colon cancer cells and improves the tumor immune microenvironment by inhibiting the proliferation of CAFs and the secretion of TGF-β. Curcumin and anti-PD-1 treatment have synergistic inhibitory effects on colon cancer.

TAK1 Promotes an Immunosuppressive Tumor Microenvironment through Cancer-Associated Fibroblast Phenotypic Conversion in Pancreatic Ductal Adenocarcinoma.

We aim to clarify the precise function of Transformed growth factor-beta 1 activated kinase-1 (TAK1) in cancer-associated fibroblasts (CAFs) within human pancreatic ductal adenocarcinoma (PDAC) by investigating its role in cytokine-mediated signaling pathways. The expression of TAK1 in pancreatic cancer was confirmed by TCGA data and human pancreatic cancer specimens. CAFs from freshly resected PDAC specimens were cultured and used in a three-dimensional model for direct and indirect co-culture with PDAC tumors to investigate TAK1 function. Additionally, organoids from KPC (LSL-K-RasLSLG12D/+; LSL-p53R172H/+; Pdx1-Cre) mice were mixed with CAFs and injected subcutaneously into C57BL/6 mice to explore in vivo functional interactions of TAK1. TCGA data revealed significant upregulation of TAK1 in PDAC, associating with a positive correlation with the T-cell exhaustion signature. Knockdown of TAK1 in CAFs decreased the iCAF signature and increased the myCAF signature both in vitro and in vivo. The absence of TAK1 hindered CAF proliferation, blocked several inflammatory factors via multiple pathways associated with immunosuppression, and hindered EMT, outgrowth in vitro in spheroid co-cultures with PDAC cells. Additionally, TAK1 inhibitor restrained tumor growth, increased CD4+ and CD8+ T cell abundance, and reduced immunosuppressive cells present in vivo. Blocking the TAK1+CAF phenotype leads to the conversion of protumorigenic CAFs to antitumorigenic CAFs. This highlights TAK1 as a potential therapeutic target, particularly in CAFs, and represents a novel avenue for combined immunotherapy in PDAC.

FHL2 expression by cancer-associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma.

Cancer-associated fibroblasts (CAFs) contribute to the progression of lung cancer. Four and a half LIM domain protein-2 (FHL2) is a component of focal adhesion structures. We analyzed the function of FHL2 expressed by CAFs in lung adenocarcinoma. Expression of FHL2 in fibroblast subtypes was investigated using database of single-cell RNA-sequencing of lung cancer tissue. The role of FHL2 in the proliferation and migration of CAFs was assessed. The effects of FHL2 knockout on the migration and invasion of human lung adenocarcinoma cells and tube formation of endothelial cells induced by CAF-conditioned medium (CM) were evaluated. The effect of FHL2 knockout in CAFs on metastasis was determined using a murine orthotopic lung cancer model. The prognostic significance of stromal FHL2 was assessed by immunohistochemistry in human adenocarcinoma specimens. FHL2 is highly expressed in myofibroblasts in cancer tissue. TGF-β1 upregulated FHL2 expression in CAFs and FHL2 knockdown attenuated CAF proliferation. FHL2 knockout reduced CAF induced migration of A110L and H23 human lung adenocarcinoma cell lines, and the induction of tube formation of endothelial cells. FHL2 knockout reduced CAF-induced metastasis of lung adenocarcinomas in an orthotopic model in vivo. The concentration of Osteopontin (OPN) in CM from CAF was downregulated by FHL2 knockout. siRNA silencing and antibody blocking of OPN reduced the pro-migratory effect of CM from CAF on lung cancer cells. In resected lung adenocarcinoma specimens, positive stromal FHL2 expression was significantly associated with higher microvascular density and worse prognosis. In conclusion, FHL2 expression by CAFs enhances the progression of lung adenocarcinoma by promoting angiogenesis and metastasis.

LncRNAPVT1 is Associated with Cancer-Associated Fibroblasts Proliferation Through Regulating TGF-βin Oral Squamous Cell Carcinoma

ABSTRACT Introduction Human oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and has a poor survival rate. Cell-cell communication between OSCC cells and cancer-associated fibroblasts (CAFs) plays important roles in OSCC progression. We previously demonstrated that CAFs promote OSCC cell migration and invasion. However, how OSCC cells influence CAFs proliferation is unknown. Methods Knockdown of PVT1 was confirmed using lentivirus infection technique. CAFs in tissues were identified by staining the cells with α-SMA using immunohistochemical technique. CCK-8 assay was used to evaluate cell proliferation. The mRNA level of a gene was measured by qRT-PCR. Secreted TGF-β were detected using ELISA assay. Results We found that knockdown of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was associated with a low density of CAFs in xenograft tumors in mice; further analysis revealed that PVT1 in OSCC cells induced CAF proliferation through transforming growth factor (TGF)-β. Discussion Our results demonstrate that lncRNA PVT1 in tumor cells participates in CAF development in OSCC by regulating TGF-β. This study revealed a new mechanism by which PVT1 regulates OSCC progression and PVT1 is a potential therapeutic target in OSCC

Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

BackgroundThe proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs.MethodsWe investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models.ResultsHigh infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs.ConclusionOur data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.

Synergistic antitumor activity of METTL3 peptide degrader and immune checkpoint inhibitor in advanced urothelial cancer.

e14623 Background: In patients afflicted with advanced urothelial carcinoma (aUC), the durable response rate to immune checkpoint inhibitor (ICI) is approximately 20%. Recent study showed the pivotal role of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) in immune escape and the progression of aUC. This study aimed to develop a novel METTL3 peptide degrader RKL and investigate its synergistic effects and underlying mechanisms in combination with ICI for aUC. Methods: Methyltransferase expression profiles were scrutinized using The Cancer Genome Atlas database; while the prognostic implications of METTL3 expression in response to ICI were analyzed in the IMvigor210 cohort study. Single-cell RNA sequencing (scRNA-seq) was employed to elucidate the mechanistic insights. The METTL3 peptide degrader RKL was synthesized using Fmoc solid-phase peptide synthesis and purified with high-performance liquid chromatography. In vivo and in vitro experiments were conducted to assess the impact of RKL, ICI, and their combination on aUC inhibition. Results: In aUC, METTL3 exhibited the most significant up-regulation among m6A methyltransferases, while METTL14 and WTAP showed down-regulated expression. In the IMvigor210 cohort study, aUC patients with elevated METTL3 expression and treated with ICI demonstrated a poorer prognosis. scRNA-seq revealed that METTL3 upregulation promoted the expression of genes regulating cancer-associated fibroblast proliferation, contributing to enhanced tumor resistance to immunotherapy. RKL demonstrated high toxicity to urothelial carcinoma cell lines, inhibiting their proliferative, migratory, and invasive capacities in vitro. The combined treatment of RKL and ICI significantly reduced tumor volume (P<0.001), with a complete response achieved in 87.5% (7/8) of mice in the combination group, 12.5% (1/8) in the RKL group, and 25.0% (2/8) in the ICI group. Conclusions: METTL3 overexpression in aUC is associated with immune escape and resistance to ICI therapy. The METTL3 peptide degrader RKL, when combined with ICI, demonstrates synergistic antitumor effects in aUC, warranting further exploration in clinical research.

Anlotinib may enhance the efficacy of anti-PD1 therapy by inhibiting the AKT pathway and promoting the apoptosis of CAFs in lung adenocarcinoma

Although PD-1 inhibitors have revolutionized the treatment paradigm of non-small cell lung cancer (NSCLC), their efficacy in treating NSCLC has remained unsatisfactory. Targeting cancer-associated fibroblasts (CAFs) is a potential approach for improving the immunotherapy response. Multitarget antiangiogenic tyrosine kinase receptor inhibitors (TKIs) can enhance the efficacy of PD-1 inhibitors in NSCLC patients. However, the effects and mechanisms of antiangiogenic TKIs on CAFs have not been elucidated. In this study, we first compared anlotinib with other antiangiogenic TKIs and confirmed the superior efficacy of anlotinib. Furthermore, we established NSCLC-associated CAF models and found that anlotinib impaired CAF viability and migration capacity and contributed to CAF apoptosis and cell cycle arrest in the G2/M phase. Moreover, anlotinib treatment attenuated the capacity of CAFs to recruit lung cancer cells and macrophages. Experiments in animal models suggested that anlotinib could enhance the efficacy of anti-PD1 therapy in NSCLC and affect CAF proliferation and apoptosis. Anlotinib increased the abundance of tumor-infiltrating CD8 + T cells, and PD-1 inhibitor-induced cytotoxicity to tumor cells was achieved through the transformation of the tumor microenvironment (TME) caused by anlotinib, which may partly explain the synergistic antitumor effect of anlotinib and PD-1 inhibitors. Mechanistically, anlotinib affects CAF apoptosis and cell viability at least in part by inhibiting the AKT pathway. In conclusion, our study suggested that anlotinib could regulate the TME, inhibit the AKT pathway and promote CAF apoptosis, providing new insights into the antitumor effect of anlotinib and improving the efficacy of immunotherapy.

Stabilizing Tumor-Resident Mast Cells Restores T-Cell Infiltration and Sensitizes Sarcomas to PD-L1 Inhibition.

To explore the cellular cross-talk of tumor-resident mast cells (MC) in controlling the activity of cancer-associated fibroblasts (CAF) to overcome tumor microenvironment (TME) abnormalities, enhancing the efficacy of immune-checkpoint inhibitors in sarcoma. We used a coculture system followed by further validation in mouse models of fibrosarcoma and osteosarcoma with or without administration of the MC stabilizer and antihistamine ketotifen. To evaluate the contribution of ketotifen in sensitizing tumors to therapy, we performed combination studies with doxorubicin chemotherapy and anti-PD-L1 (B7-H1, clone 10F.9G2) treatment. We investigated the ability of ketotifen to modulate the TME in human sarcomas in the context of a repurposed phase II clinical trial. Inhibition of MC activation with ketotifen successfully suppressed CAF proliferation and stiffness of the extracellular matrix accompanied by an increase in vessel perfusion in fibrosarcoma and osteosarcoma as indicated by ultrasound shear wave elastography imaging. The improved tissue oxygenation increased the efficacy of chemoimmunotherapy, supported by enhanced T-cell infiltration and acquisition of tumor antigen-specific memory. Importantly, the effect of ketotifen in reducing tumor stiffness was further validated in sarcoma patients, highlighting its translational potential. Our study suggests the targeting of MCs with clinically administered drugs, such as antihistamines, as a promising approach to overcome resistance to immunotherapy in sarcomas.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect

BackgroundGemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PurposeTo investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. MethodsPC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. ResultsCAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. ConclusionThese results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Exosomes derived from ovarian cancer cells regulate proliferation and migration of cancer-associated fibroblasts

Cancer-associated fibroblast (CAF) is an essential risk factor for ovarian cancer. Exosomes can mediate cellular communication in the tumour microenvironment, but the interaction of tumour cell exosomes with CAF is less studied in Ovarian cancer. This study identified H19/miR-29c-3p/LOXL2-COL1A1 as a ceRNA regulatory network involved in regulating tumour matrix-associated signaling pathways associated with CAF. Cellular assays demonstrated that exosomes from ovarian cancer cell line SKOV3 significantly promoted the proliferation and migration of CAF. The results of mixed transplantation tumour experiments in nude mice showed that exosomes of SKOV3 significantly promoted tumour growth. Ovarian cancer tumour-derived exosomes can regulate CAF proliferation and migration through H19/miR-29c-3p/LOXL2-COL1A1. This study reveals the regulatory role of tumour exosomes on CAF, which may provide a theoretical basis for the development of therapeutic regimens targeting fibroblasts in ovarian cancer.

Abstract 2502: The role of high mobility group box-1 protein (HMGB1) in the microenvironment of non-small cell lung cancer

Abstract Introduction: The tumor microenvironment (TME) is critical in determining tumor development and progression. Among stromal cells in TME, cancer associated fibroblasts (CAFs) play a central role and are involved in various signals through their interaction with cancer cells in most solid tumors, leading to cancer progression and acquisition of drug resistance. Under these TME conditions, tumor cell death such as necrosis occurs, which in turn, results in the ectopic release of bioactive molecules collectively termed as damage-associated molecular patterns (DAMPs), which has been reported to play an important role in cancer progression. The purpose of this study is to clarify the effects of cancer cell-derived HMGB1, a representative DAMPs, on CAFs and to investigate whether it could be a potential therapeutic target. Methods: We established CAFs from resected non-small cell lung cancer (NSCLC) specimens and investigated the biological activity of HMGB1 on CAFs using in vitro co-culture models with NSCLC cell lines. Furthermore, we assessed the therapeutic efficacy of targeting cancer cell-derived HMGB1 using anti-HMGB1 neutralizing antibody. Results: Intracellular HMGB1 was expressed more in NSCLC cells than in normal bronchial epithelial cells and CAFs, and released from cancer cells extracellularly upon treatment with anticancer drugs. The stimulation with recombinant HMGB1 promoted the proliferation of CAFs in a dose-dependent manner, but not lung cancer cells. Co-culture with CAFs promoted cancer cells’ migratory ability and drug resistance, and stimulation with recombinant HMGB1 further enhanced these promoting effects, while administration of anti-HMGB1 neutralizing antibody suppressed these effects. On the other hand, these inhibitory effects were not observed in the absence of CAFs, suggestingHMGB1 has effect on CAFs, but not direct on cancer cells. Conclusion: HMGB1 plays an important role in cancer progression and acquisition of therapeutic resistance by upregulating the CAFs’ proliferation and activity and could be a therapeutic target in NSCLC. Citation Format: Tomohiro Habu, Keiichi Date, Ken Suzawa, Mao Yoshikawa, Masayoshi Ohki, Kazuma Iwata, Naoki Matsuda, Yin Min Thu, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. The role of high mobility group box-1 protein (HMGB1) in the microenvironment of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2502.

Abstract 5832: Integrinα5 mediates the direct heterocellular interaction between cancer cells and cancer-associated fibroblasts and promotes peritoneal metastasis of diffuse-type gastric carcinoma

Abstract Diffuse-type gastric carcinoma (DGC) is an aggressive subtype of gastric cancer characterized by rapid infiltrative growth and frequent peritoneal metastasis. Peritoneal metastasis is the main cause of poor prognosis and decreased quality of life for patients with DGC. Histologically, DGC tumor lesions are associated with massive fibrotic stroma caused by the extensive proliferation of cancer-associated fibroblasts (CAFs). Peritoneally metastasized tumors of DGC are also accompanied by abundant fibrotic stroma and CAFs. CAFs are known to promote the progression of DGC by enhancing the growth, invasion, and dissemination of tumor cells. Previously, we reported that the direct heterocellular interaction between cancer cells and CAFs is necessary for the invasion and peritoneal metastasis of DGC. In this study, we aimed to identify and target the molecules that mediate such direct heterocellular interaction. Monoclonal antibodies (mAbs) against intact DGC cells were generated and subjected to high-throughput screening to obtain mAbs that block the adhesion of DGC cells to CAFs. As a result, several such inhibitory mAbs were obtained. Immunoprecipitation and mass spectrometry revealed that all mAbs recognized integrin α5 complexed with integrin β1. Blocking integrin α5 in DGC cells abrogated the adhesion of DGC cells to CAFs. Similarly, blocking fibronectin, a ligand of integrin α5β1, deposited on CAFs blocked the heterocellular adhesion. Furthermore, administration of mAbs or knockout of integrin α5 in DGC cells suppressed their invasion led by CAFs in vitro and peritoneal metastasis in a mouse xenograft model. Analysis of the public database revealed that integrin α5 and fibronectin were highly expressed in DGC. In addition, integrin α5 and fibronectin expression levels were correlated with worse overall survival in DGC patients. Taken together, these results show that integrin α5 mediates the heterotypic cancer cell-CAF interaction during peritoneal metastasis of DGC and may thus be a promising therapeutic target. Citation Format: Hideki Yamaguchi, Shingo Miyamoto, Yoshiko Nagano, Makoto Miyazaki, Yuko Nagamura. Integrinα5 mediates the direct heterocellular interaction between cancer cells and cancer-associated fibroblasts and promotes peritoneal metastasis of diffuse-type gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5832.

Relaxin-encapsulated polymeric metformin nanoparticles remodel tumor immune microenvironment by reducing CAFs for efficient triple-negative breast cancer immunotherapy

Cancer-associated fibroblasts (CAFs) are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment, leading to the failure of triple-negative breast cancer (TNBC) immunotherapy. Therefore, depleting CAFs may enhance the effect of immunotherapy (such as PD-L1 antibody). Relaxin (RLN) has been demonstrated to significantly improve transforming growth factor-β (TGF-β) induced CAFs activation and tumor immunosuppressive microenvironment. However, the short half-life and systemic vasodilation of RLN limit its in vivo efficacy. Here, plasmid encoding relaxin (pRLN) to locally express RLN was delivered with a new positively charged polymer named polymeric metformin (PolyMet), which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before. In order to improve the stability of pRLN in vivo, this complex was further formed lipid poly-γ-glutamic acid (PGA)/PolyMet-pRLN nanoparticle (LPPR). The particle size of LPPR was 205.5 ± 2.9 nm, and the zeta potential was +55.4 ± 1.6 mV. LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro. In vivo, it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment, which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration. Thus, LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse, and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody (aPD-L1). Altogether, this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.

Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway

Cancer-associated fibroblasts (CAFs) are an integral part of the tumor microenvironment (TME), which is involved in therapy resistance. This study aimed to investigate the role of CAFs in radiosensitivity of breast cancer cells. The CAFs were isolated from the breast cancer tissues, and the conditioned medium was collected to culture breast cancer cells. Radiation-induced DNA damage was evaluated by immunofluorescence and western blotting. Cytokine array and enzyme-linked immunosorbent assay were performed to analyze the secretion of cytokines and growth factors. An in vitro clonogenic survival assay and in vivo xenograft mouse model were performed to determine the radiosensitivity of breast cancer cells. Finally, the expression of hepatocyte growth factor (HGF) and c-Met in the breast cancer tissues were detected by immunohistochemistry. The CAFs were found to secrete HGF to activate the c-Met signaling pathway, which induced epithelial-to-mesenchymal transition, growth, and radioresistance of breast cancer cells. Furthermore, radiation was observed to enhance HGF secretion by CAFs and increase c-Met expression in breast cancer cells, which led to HGF/c-Met signaling pathway activation. Moreover, radiation-induced tumor necrosis factor α (TNFα) secretion by breast cancer cells promoted CAF proliferation and HGF secretion. Additionally, HGF and c-Met high expression were associated with worse recurrence-free survival in patients with breast cancer who had received radiation therapy. The findings revealed that HGF and TNFα are critical for the crosstalk between breast cancer cells and CAFs in the TME and that the HGF/c-Met signaling pathway is a promising therapeutic target for radiosensitizing breast cancer.

Abstract C068: Isorhamnetin restricts cancer-associated fibroblasts (CAFs) phenotypic plasticity, poising them towards tumor-restraining myCAFs: Potential use of isorhamnetin as neoadjuvant in pancreatic ductal adenocarcinoma (PDAC)

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer due lack of early diagnosis and inherent refractory to traditional chemo- and even novel immune-therapies. PDAC is marked by its extensive desmoplastic stroma containing extracellular matrix (ECM), vasculature and most importantly cancer-associated fibroblasts (CAFs). CAFs that exist in heterogenous subtypes are responsible for shaping PDAC tumor microenvironment being the main source of: ECM deposition leading up to desmoplasia that hampers the efficacy of various therapeutics, as well as diverse cytokines and growth factors that promote the growth of cancer cells and the shift towards dedifferentiated and invasive phenotype and contribute towards immune suppressive microenvironment. Thus, modulating CAFs phenotype is an attractive mean to increase the efficacy of therapies targeting PDAC cells. Here we assessed the potential therapeutic benefits of Isorhamnetin, a flavonoid previously shown to exert anti-proliferative, anti-fibrotic and anti-inflammatory effect in various cancer types, to modulates PDAC microenvironment by characterizing Isorhamnetin effect on human PDAC-derived CAFs. Isorhamnetin inhibited CAFs proliferation, induced apoptosis, and cell cycle arrest by impairing mitochondrial health. Importantly, isorhamnetin attenuates the expression of inflammatory CAFs (iCAFs) signature including IL1A, IL6, LIF, CXCL1 and promotes phenotype shift towards myofibroblasts (myCAFs)-as evident in higher level of αSMA positive CAFs. Our results demonstrate antiproliferative effect of isorhamnetin on human PDAC-derived CAFs and a phenotype switch from iCAFs to myCAFs and warrant further investigation for its use in combinatorial therapy that target both the cancer and the tumor microenvironment as a whole. Citation Format: Munkhzul Ganbold, Pakavarin Louphrasitthiphol, Yoshihiro Miyazaki, Tatsuya Oda, Kenichi Tominaga, Hiroko Isoda. Isorhamnetin restricts cancer-associated fibroblasts (CAFs) phenotypic plasticity, poising them towards tumor-restraining myCAFs: Potential use of isorhamnetin as neoadjuvant in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C068.

Cell Markers Present in the TGF-β-Activated Transdifferentiation of Normal Fibroblasts to Cancer-Associated Fibroblast in Endometrial Cancer

Introduction: Cancer-associated fibroblasts (CAFs) are an essential component of carcinogenesis. The biological origins of CAFs in humans depend on the histotype of the tumour and the region where it first originated, and thus CAFs could be derived from many different cell types. Normal fibroblasts (NFs) are abundant in the endometrium and are highly susceptible to transdifferentiation to CAFs through TGF-β activation. This study aims to identify the cell markers present in the TGF-β signalling pathways for the transition of NFs to CAFs in endometrial cancer (EC). Methods: EC will be chemically induced in ICR (Institute of Cancer Research) mice with N-methyl-N-nitrosourea (MNU) and a 17β-estradiol (E2) diet. Cancer progression will be monitored using magnetic resonance imaging (MRI) at a field of 4.7 T. CAFs will then be isolated from the TME using PDGFRα as the cell marker. Immunohistochemistry (IHC) staining will be used on EC tumour cells to identity the presence the location of cell markers phosphorylated Smad2/3 (pSmad2/3), ERK1/2, and PI3K. Anticipated Results: CAF cells are expected to test positive for markers expressed in PDGFRα mediated signalling pathways. Presence of pSmad2/3 is expected to increase over time as usage of the canonical pathway increases in CAF establishment and cancer progression. Non-canonical pathway activation would show levels of ERK2/3 and PI3K. Discussion: pSmad2/3 levels will be examined to determine the usage of the canonical pathway in CAF expansion. Detection of pSmad 2/3 or PI3K/ERK2/3 allows for targeted therapy on the appropriate TGF-ß pathway to block CAF production, thus stopping tumour progression. Suppression of the pathways by targeting specific biomarkers such as PTEN to inhibit mTOR or CAV-1 inhibitors could normalize an upregulated or downregulated TGF-ß pathway. Conclusion: Identifying the key cell markers in the transdifferentiation of NFs allows for the targeting of specific proteins that play a role in the signalling pathways. Standardizing identification of significant cell markers in CAF establishment improves individualized treatment to the cancer patient. Treatment(s) would target the cell markers involved to prevent further CAF proliferation and tumour development

Tumor microenvironmental 15-PGDH depletion promotes fibrotic tumor formation and angiogenesis in pancreatic cancer.

The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E2 (PGE2). Although inhibition of 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) is reported to lead to PGE2 accumulation, the role of 15‐PGDH expression in the tumor microenvironment remains unclear. We utilized Panc02 murine pancreatic cancer cells for orthotopic transplantation into wild‐type and 15‐pgdh+/− mice and found that 15‐pgdh depletion in the tumor microenvironment leads to enhanced tumorigenesis accompanied by an increase in cancer‐associated fibroblasts (CAFs) and the promotion of fibrosis. The fibrotic tumor microenvironment is widely considered to be hypovascular; however, we found that the angiogenesis level is maintained in 15‐pgdh+/− mice, and these changes were also observed in a genetically engineered PDAC mouse model. Further confirmation revealed that fibroblast growth factor 1 (FGF1) is secreted by pancreatic cancer cells after PGE2 stimulation, consequently promoting CAF proliferation and vascular endothelial growth factor A (VEGFA) expression in the tumor microenvironment. Finally, in 15‐pgdh+/−Acta2‐TK mice, depletion of fibroblasts inhibited angiogenesis and cancer cell viability in orthotopically transplanted tumors. These findings highlighted the role of 15‐pgdh downregulation in enhancing PGE2 accumulation in the pancreatic tumor microenvironment and in subsequently maintaining the angiogenesis level in fibrotic tumors along with CAF expansion.

A Mouse Model of Damp-Heat Syndrome in Traditional Chinese Medicine and Its Impact on Pancreatic Tumor Growth.

BackgroundDamp-heat syndrome is one of the most important syndrome types in the traditional Chinese medicine (TCM) syndrome differentiation and treatment system, as well as the core pathogenesis of pancreatic cancer (PC) which remains a challenge to medical researchers due to its insidious onset and poor prognosis. Great attention has been given to the impact of damp-heat syndrome on tumorigenesis and progression, but less attention has been given to damp-heat modeling per se. Studying PC in a proper damp-heat syndrome animal model can recapitulate the actual pathological process and contribute to treatment strategy improvement.MethodsHere, an optimized damp-heat syndrome mouse model was established based on our prior experience. The Fibonacci method was applied to determine the maximum tolerated dosage of alcohol for mice. Damp-heat syndrome modeling with the old and new methods was performed in parallel of comparative study about general appearance, food intake, water consumption and survival. Major organs, including the liver, kidneys, lungs, pancreas, spleen, intestines and testes, were collected for histological evaluation. Complete blood counts and biochemical tests were conducted to characterize changes in blood circulation. PC cells were subcutaneously inoculated into mice with damp-heat syndrome to explore the impact of damp-heat syndrome on PC growth. Hematoxylin-eosin staining, Masson staining and immunohistochemistry were performed for pathological evaluation. A chemokine microarray was applied to screen the cytokines mediating the proliferation-promoting effects of damp-heat syndrome, and quantitative polymerase chain reaction and Western blotting were conducted for results validation.ResultsThe new modeling method has the advantages of mouse-friendly features, easily accessible materials, simple operation, and good stability. More importantly, a set of systematic indicators was proposed for model evaluation. The new modeling method verified the pancreatic tumor-promoting role of damp-heat syndrome. Damp-heat syndrome induced the proliferation of cancer-associated fibroblasts and promoted desmoplasia. In addition, circulating and tumor-located chemokine levels were altered by damp-heat syndrome, characterized by tumor promotion and immune suppression.ConclusionsThis study established a stable and reproducible murine model of damp-heat syndrome in TCM with systematic evaluation methods. Cancer associated fibroblast-mediated desmoplasia and chemokine production contribute to the tumor-promoting effect of damp-heat syndrome on PC.

Arecoline Is Associated With Inhibition of Cuproptosis and Proliferation of Cancer-Associated Fibroblasts in Oral Squamous Cell Carcinoma: A Potential Mechanism for Tumor Metastasis.

BackgroundMetastatic disease remains the primary cause of death in patients with oral squamous cell carcinoma (OSCC), especially those who use betel nut. The different steps of the metastatic cascade rely on reciprocal interactions between cancer cells and the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are regarded as a significant component in the TME of OSCC. However, the precise mechanisms regulating CAFs in OSCC are poorly understood.MethodsThirteen genes related to the arecoline were analyzed to explore the significant ones involved in arecoline-related OSCC metastasis. The GSE139869 (n = 10) and The Cancer Genome Atlas (TCGA)-OSCC data (n = 361) were mined for the identification of the differentially expressed genes. The least absolute shrinkage and selection operator (LASSO) regression was performed to identify the independent prognostic signatures. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to explore the functional enrichment of selected genes, and gene set enrichment analysis of cuproptosis-related genes was completed. Spearman’s analysis and Tumor Immune Estimation Resource (TIMER) were used to visualize the correlation between the infiltration of CAFs and the gene expression. The correlation analysis of the cells and different genes, including CAF infiltration and transcripts per million expression, was assessed. The relationship between arecoline and CAFs was confirmed by cell counting kit-8 assay (CCK-8). CancerSEA was searched to identify the single-cell phenotype.ResultArecoline-associated fibrosis-related OSCC differentially expressed genes (AFOC-DEGs), namely, PLAU, IL1A, SPP1, CCL11, TERT, and COL1A2, were screened out and selected from the Gene Expression Omnibus (GEO) database and TCGA database. AFOC-DEGs were highly expressed in OSCC, which led to poor survival of patients. Functional enrichment analysis, protein–protein interaction network construction, and Spearman’s correlation analysis all suggested that AFOC-DEGs were closely associated with cuproptosis. Cellular experiments demonstrated that arecoline stimulation could significantly increase the cell viability of CAFs. Single-sample Gene Set Enrichment Analysis (ssGSEA) results showed that GLS and MTF1 were highly expressed when fibroblasts proliferated at high enrichment levels. In addition, analysis of single-cell sequencing results suggested that OSCC cells with high expression of AFOC-DEGs were associated with OSCC metastasis.ConclusionWe found a close association between arecoline, cuproptosis, and CAFs, which might play an important role in the metastasis of OSCC.