Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis.

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5'-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

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.

Co-targeting FAK and Gli1 inhibits the tumor-associated macrophages-released CCL22-mediated esophageal squamous cell carcinoma malignancy.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.

PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy.

Stroma surrounding the tumor cells plays crucial roles for tumor progression. However, little is known about the factors that maintain the symbiosis between stroma and tumor cells. In this study, we found that the transcriptional regulator-signal transducer and activator of transcription 3 (Stat3) was frequently activated in cancer-associated fibroblasts (CAFs), which was a potent facilitator of tumor malignancy, and formed forward feedback loop with platelet-activating factor receptor (PAFR) both in CAFs and tumor cells. Importantly, PAFR/Stat3 axis connected intercellular signaling crosstalk between CAFs and cancer cells and drove mutual transcriptional programming of these two types of cells. Two central Stat3-related cytokine signaling molecules-interleukin 6 (IL-6) and IL-11 played the critical role in the process of PAFR/Stat3 axis-mediated communication between tumor and CAFs. Pharmacological inhibition of PAFR and Stat3 activities effectively reduced tumor progression using CAFs/tumor co-culture xenograft model. Our study reveals that PAFR/Stat3 axis enhances the interaction between tumor and its associated stroma and suggests that targeting this axis can be an effective therapeutic strategy against tumor malignancy.

Src heterodimerically activates Lyn or Fyn to serve as targets for the diagnosis and treatment of esophageal squamous cell carcinoma.

Although Src is one of the oldest and most investigated oncoproteins, its function in tumor malignancy remains to be defined further. In this study, we demonstrated that the inhibition of Src activity by ponatinib effectively suppressed several malignant phenotypes of esophageal squamous cell carcinoma (ESCC) both in vitro and in vivo, whereas it did not produce growth-inhibitory effects on normal esophageal epithelial cells (NEECs). Importantly, we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src interacted with the members of Src-family kinases (SFKs), such as Fyn or Lyn, to form heterodimers. Src interactions with Fyn and Lyn phosphorylated the tyrosine sites in SH2 (Fyn Tyr185 or Lyn Tyr183) and kinase domains (Fyn Tyr420 or Lyn Tyr397), which critically contributed to ESCC development. By contrast, Src could not form heterodimers with Fyn or Lyn in NEECs. We used RNA sequencing to comprehensively demonstrate that the inhibition of Src activity effectively blocked several critical tumor-promoting pathways, such as JAK/STAT, mTOR, stemness-related, and metabolism-related pathways. Results of the real-time polymerase chain reaction (RT-PCR) assay confirmed that Lyn and Fyn were critical effectors for the Src-mediated expression of tumor growth or metastasis-related molecules. Furthermore, results of the clinical ESCC samples showed that the hyperactivation of pSrc Tyr419, Fyn Tyr185 or Tyr420, and Lyn Tyr183 or Tyr397 could be biomarkers of ESCC prognosis. This study illustrates that Src/Fyn and Src/Lyn heterodimers serve as targets for the treatment of ESCC.

Tumor-associated macrophage (TAM)-derived CCL22 induces FAK addiction in esophageal squamous cell carcinoma (ESCC).

Tumor cell dependence on activated oncogenes is considered a therapeutic target, but protumorigenic microenvironment-mediated cellular addiction to specific oncogenic signaling molecules remains to be further defined. Here, we showed that tumor-associated macrophages (TAMs) produced an abundance of C-C motif chemokine 22 (CCL22), whose expression in the tumor stroma was positively associated with the level of intratumoral phospho-focal adhesion kinase (pFAK Tyr397), tumor metastasis and reduced patient survival. Functionally, CCL22-stimulated hyperactivation of FAK was correlated with increased malignant progression of cancer cells. CCL22-induced addiction to FAK was demonstrated by the persistent suppression of tumor progression upon FAK-specific inhibition. Mechanistically, we identified that diacylglycerol kinase α (DGKα) acted as a signaling adaptor to link the CCL22 receptor C-C motif chemokine receptor 4 (CCR4) and FAK and promoted CCL22-induced activation of the FAK/AKT pathway. CCL22/CCR4 signaling activated the intracellular Ca2+/phospholipase C-γ1 (PLC-γ1) axis to stimulate the phosphorylation of DGKα at a tyrosine residue (Tyr335) and promoted the translocation of DGKα to the plasma membrane to assemble the DGKα/FAK signalosome, which critically contributed to regulating sensitivity to FAK inhibitors in cancer cells. The identification of TAM-driven intratumoral FAK addiction provides opportunities for utilizing the tumor-promoting microenvironment to achieve striking anticancer effects.

A Comparative Study of the Effects of Different Decellularization Methods and Genipin-Cross-Linking on the Properties of Tracheal Matrices.

BACKGROUND: Different decellularization methods can affect the integrity and the biomechanical and biocompatible properties of the tracheal matrix. Natural cross-linking with genipin can be applied to improve those properties. The goals of this study were to evaluate the effects of different decellularization methods on the properties of genipin-cross-linked decellularized tracheal matrices in rabbits. METHODS: The tracheas of New Zealand rabbits were decellularized by the Triton-X 100-processed method (TPM) and the detergent-enzymatic method (DEM) and were then cross-linked with genipin. Mechanical tests, haematoxylin-eosin staining, Masson trichrome staining, Safranin O staining, DAPI staining, scanning electronic microscopy (SEM), and biocompatibility tests were used to evaluate the treatment. The bioengineered trachea and control trachea were then implanted into allogeneic rabbits for 30 days. The structural and functional analyses were performed after transplantation. RESULTS: The biomechanical tests demonstrated that the biomechanical properties of the decellularized tracheas decreased and that genipin improved them (p < 0.05). The histological staining results revealed that most of the mucosal epithelial cells were removed and that the decellularized trachea had lower immunogenicity than the control group. The analysis of SEM revealed that the decellularized trachea retained the micro- and ultra-structural architectures of the trachea and that the matrices cross-linked with genipin were denser. The biocompatibility evaluation and in vivo implantation experiments showed that the decellularized trachea treated with the DEM had better biocompatibility than that treated with the TPM and that immunogenicity in the cross-linked tissues was lower than that in the uncross-linked tissues (p < 0.05). CONCLUSIONS: Compared with the trachea treated with the TPM, the rabbit trachea processed by the DEM had better biocompatibility and lower immunogenicity, and its structural and mechanical characteristics were effectively improved after the genipin treatment, which is suitable for engineering replacement tracheal tissue.

Selection of the optimum 3D-printed pore and the surface modification techniques for tissue engineering tracheal scaffold in vivo reconstruction.

The influences of pore sizes and surface modifications on biomechanical properties and biocompatibility (BC) of porous tracheal scaffolds (PTSs) fabricated by polycaprolactone (PCL) using 3D printing technology. The porous grafts were surface-modified through hydrolysis, amination, and nanocrystallization treatment. The surface properties of the modified grafts were characterized by energy dispersive spectroscopy (EDS) and scanning electron microscopy (SEM). The materials were cocultured with bone marrow mesenchymal stem cells (BMSCs). The effect of different pore sizes and surface modifications on the cell proliferation behavior was evaluated by the cell counting kit-8 (CCK-8). Compared to native tracheas, the PTS has good biomechanical properties. A pore diameter of 200 µm is the optimum for cell adhesion, and the surface modifications successfully improved the cytotropism of the PTS. Allogeneic implantation confirmed that it largely retains its structural integrity in the host, and the immune rejection reaction of the PTS decreased significantly after the acute phase. Nano-silicon dioxide (NSD)-modified PTS is a promising material for tissue engineering tracheal reconstruction. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 360-370, 2019.