Polarization Of Tumor-associated Macrophages Research Articles

Targeting both death and paracaspase domains of MALT1 with antisense oligonucleotides overcomes resistance to immune-checkpoint inhibitors.

Targeting MALT1's paracaspase activity has been explored for B cell lymphoma and solid tumors. While the role of MALT1 in promoting cancer cell proliferation has been investigated, its involvement in immune evasion is unclear. Here we report that MALT1 promotes immune evasion through its paracaspase and death domain. In a paracaspase-dependent manner, MALT1 protects CD274 mRNA from degradation by its cleavage of ROQUIN1 and ROQUIN2. In a death-domain-dependent manner, MALT1 promotes the proliferation and polarization of tumor-associated macrophages to generate an immunosuppressive tumor microenvironment. Targeting MALT1 with antisense oligonucleotides inhibits PD-L1 expression in patient-derived tumor cells and suppresses the proliferation and M2-like polarization of tumor-associated macrophages isolated from patients with cancer. In preclinical models of solid tumors in female mice, treatment with MALT1 antisense oligonucleotides overcomes resistance to immune-checkpoint inhibitors. Together, our study demonstrates that targeting MALT1 is a potential strategy to overcome immune-checkpoint inhibitor resistance.

Multi-omics analysis revealed the novel role of NQO1 in microenvironment, prognosis and immunotherapy of hepatocellular carcinoma

NAD(P)H dehydrogenase quinone 1 (NQO1) is overexpressed in various cancers and is strongly associated with an immunosuppressive microenvironment and poor prognosis. In this study, we explored the role of NQO1 in the microenvironment, prognosis and immunotherapy of Hepatocellular carcinoma (HCC) using multi-omics analysis and machine learning. The results revealed that NQO1 was significantly overexpressed in HCC cells. NQO1+HCC cells were correlated with poor prognosis and facilitated tumor-associated macrophages (TAMs) polarization to M2 macrophages. We identified core NQO1-related genes (NRGs) and developed the NRGs-related risk-scores in hepatocellular carcinoma (NRSHC). The comprehensive nomogram integrating NRSHC, age, and pathological tumor-node-metastasis (pTNM) Stage achieved an area under the curve (AUC) above 0.7, demonstrating its accuracy in predicting survival outcomes and immunotherapy responses of HCC patients. High-risk patients exhibited worse prognoses but greater sensitivity to immunotherapy. Additionally, a web-based prediction tool was designed to enhance clinical utility. In conclusion, NQO1 may play a critical role in M2 polarization and accelerates HCC progression. The NRSHC model and accompanying tools offer valuable insights for personalized HCC treatment.

The multifaceted role of phosphodiesterase 4 in tumor: from tumorigenesis to immunotherapy

Phosphodiesterase 4 (PDE4) is an enzyme that specifically hydrolyzes the second messenger cAMP and has a critical role in the regulation of a variety of cellular functions. In recent years, PDE4 has attracted great interest in cancer research, and its role in tumorigenesis and development has been gradually elucidated. Research indicates that abnormal expression or heightened activity of PDE4 is associated with the initiation and progression of multiple cancers, including lung, colorectal, and hematological cancers, by facilitating cell proliferation, migration, invasion, and anti-apoptosis. Moreover, PDE4 also influences the tumor immune microenvironment, significantly immune evasion by suppressing anti-tumor immune responses, reducing T-cell activation, and promoting the polarization of tumor-associated macrophages toward a pro-tumorigenic phenotype. However, the PDE4 family may have both oncogenic and tumor-suppressive effects, which could depend on the specific type and grade of the tumor. PDE4 inhibitors have garnered substantial interest as potential anti-cancer therapeutics, directly inhibiting tumor cell growth and restoring immune surveillance capabilities to enhance the clearance of tumor cells. Several PDE4 inhibitors are currently under investigation with the aim of exploring their potential in cancer therapy, particularly in combination strategies with immune checkpoint inhibitors, to improve therapeutic efficacy and mitigate the side effects of conventional chemotherapy. This review provides an overview of PDE4 in tumorigenesis, drug resistance, immunotherapy, and the anti-tumor actions of its inhibitors, intending to guide the exploration of PDE4 as a new target in tumor therapy.

Calculus bovis inhibits liver cancer via the Wnt/β-catenin pathway

In this paper, the mechanism of the Wnt/β-catenin pathway is introduced, and the process and principle of the experiment conducted by Huang et al is explained. We discussed the reliability of the conclusion that Calculus bovis (C. bovis ) inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer. We also offer suggestions for further studies of the use of C. bovis in the treatment of liver cancer.

Circulating mitochondrial DNA promotes M2 polarization of tumor associated macrophages and HCC resistance to sorafenib

Mitochondrial damage-associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA), TFAM (transcription factor A, mitochondrial), and ATP, which play crucial roles in the regulation of inflammatory environment in human diseases. However, the role of mitochondrial DAMPs in regulating tumor microenvironment (TME) remains unclear. Herein, we demonstrate that infiltration of M2-type tumor-associated macrophages (TAMs) was correlated with the resistance of hepatocellular carcinoma (HCC) to sorafenib. We found that cell-free mtDNA in the plasma was significantly increased in sorafenib-resistant HCC mice. Sorafenib induced mitochondrial dysfunction and promoted the release of mtDNA into extracellular matrix of HCC. Macrophages retook the mtDNA in the TME of HCC, activated TLR9 signaling, and promoted the activation of NF-κB and the polarization of TAMs into M2. Application of DNase I to digest mtDNA or depletion of macrophages with clodronate liposomes reduced M2 macrophage infiltration, decreased the growth of HCC, and sensitized the tumors to sorafenib. Furthermore, we showed that blocking the activation of TLR9 enhanced the therapeutic effect of sorafenib in HCC. Together, we demonstrate that sorafenib treatment leads to the release of mtDNA into TME in HCC, which in turn facilitates the polarization of TAMs into M2 macrophages through TLR9 activation and aggravates the resistance of HCC to sorafenib. Our study reveals a novel mechanism underlying circulating mtDAMPs in remodeling the HCC microenvironment by reprograming the TAMs and provides a new strategy for improving the therapeutic effect of sorafenib and overcoming its resistance in HCC.

Multidimensional applications of prussian blue-based nanoparticles in cancer immunotherapy

Immunotherapy holds notable progress in the treatment of cancer. However, the clinical therapeutic effect remains a significant challenge due to immune-related side effects, poor immunogenicity, and immunosuppressive microenvironment. Nanoparticles have emerged as a revolutionary tool to surmount these obstacles and amplify the potency of immunotherapeutic agents. Prussian blue nanoparticles (PBNPs) exhibit multi-dimensional immune function in cancer immunotherapy, including acting as a nanocarrier to deliver immunotherapeutic agents, as a photothermal agent to improve the efficacy of immunotherapy through photothermal therapy, as a nanozyme to regulate tumor microenvironment, and as an iron donor to induce immune events related to ferroptosis and tumor-associated macrophages polarization. This review focuses on the advances and applications of PBNPs in cancer immunotherapy. First, the biomedical functions of PBNPs are introduced. Then, based on the immune function of PBNPs, we systematically reviewed the multidimensional application of PBNPs in cancer immunotherapy. Finally, the challenges and future developments of PBNPs-based cancer immunotherapy are highlighted.Graphical abstract

Upstream-binding protein-1 promotes breast tumorigenesis by inducing NRG2-mediated metastasis, plasticity, and macrophage polarization.

Upstream-binding protein-1 promotes breast tumorigenesis by inducing NRG2-mediated metastasis, plasticity, and macrophage polarization.

Proguanil inhibits proliferation and migration in glioblastoma development through targeting CSF1R receptor.

Proguanil inhibits proliferation and migration in glioblastoma development through targeting CSF1R receptor.

UBE2I depletion regulated tumor-associated macrophage polarization into M1 type through reprogramming glycolysis and increases immunotherapy efficacy of anti-PD-L1 in ovarian cancer.

UBE2I depletion regulated tumor-associated macrophage polarization into M1 type through reprogramming glycolysis and increases immunotherapy efficacy of anti-PD-L1 in ovarian cancer.

CTHRC1 expresses in cancer-associated fibroblasts and is associated with resistance to anti-androgen therapy in prostate cancer.

CTHRC1 overexpresses in prostate cancer and is associated with the proliferation, invasion and migration of prostate cancer cells. However, the roles and mechanisms of CTHRC1 expression in prostate cancer prognosis and treatment outcomes remain unknown. This study aimed to explore the expression and gene function of CTHRC1 in prostate cancer, investigate the prognostic value and potential effect in the treatment of prostate cancer. Bulk and single-cell RNA sequencing analyses were used to evaluate the expression of CTHRC1 in prostate cancer. All data used in the study were obtained from publicly available sources to ensure transparency. Study enrolled 1999 cases of prostate cancer and 531 normal controls. Single-cell RNA sequencing profile included 62,995 cells from seven prostate primary tumors. CTHRC1 expression and prognosis analyses were conducted with these samples and verified by immunohistochemical staining. CIBERSORT algorithm was used to assess the tumor immune infiltrating cells based on bulk mRNA sequencing profiles. Genomics of drug sensitivity in cancer (GDSC) database was used to predict IC50 to anti-androgen therapy (ADT) drugs of the samples. CTHRC1 expressed in prostate cancer was higher than that in normal prostate tissue, and the expression increased with the progress of prostate cancer. CTHRC1 was the risk factor of progression-free interval (PFI). CTHRC1 was positively correlated with the infiltration of tumor-associated macrophages (TAMs). Myofibroblast-like cancer-associated fibroblasts (myCAFs) were the major CTHRC1 expressers in prostate cancer. TGF-β signaling activated in CTHRC1-positive myCAFs and was involved in TAMs polarization. Biological functions of myCAFs were enriched in hormone response and metabolism. CTHRC1 may regulate androgen receptor signaling through CCN2/CAV1/AR pathway. Moreover, ADT drug Bicalutamide and AZD3514 were less sensitive in the high CTHRC1 expression tumors. As a potential molecular target of ADT resistance in prostate cancer, CTHRC1 provides a new promising molecular approach for the diagnosis and treatment of prostate cancer.

Targeting mesothelin-CD24 axis repolarizes tumor-associated macrophages to potentiate PD-1 blockade therapy in high-grade serous ovarian cancer

BackgroundHigh-grade serous ovarian cancer (HGSOC) is a highly aggressive malignancy marked by an immunosuppressive tumor microenvironment that hinders effective immune responses. A key feature of this environment is the extensive...

CpG-Based Nanovaccines Enhance Ovarian Cancer Immune Response by Gbp2-Mediated Remodeling of Tumor-Associated Macrophages.

CpG oligodeoxynucleotides (CpG), as an immunoadjuvant, can facilitate the transformation of tumor-associated macrophages (TAMs)into tumoricidal M1 macrophages. However, the accumulation of free CpG in tumor tissues remains a substantial challenge. To address this, a nanovaccine (PLGA-CpG@ID8-M) is engineered by encapsulating CpG within PLGA using ID8 ovarian cancer cell membranes (ID8-M). This nanovaccine demonstrates remarkable efficacy in reprogramming TAMs in ovarian cancer and significantly extends survival in ID8-bearing mice. Notably, these findings indicate that the nanovaccine can also mitigate chemotherapy-induced immunosuppression by increasing the proportion of M1-like TAMs and reducing the expression of CD47 on tumor cells, thereby achieving a synergistic effect in tumor immunotherapy. Mechanistically, through transcriptome sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), and mass spectrometry-based proteomics, it is elucidated that the nanovaccine enhances the expression of Gbp2 and promotes the recruitment of Pin1, which activates the NFκB signaling pathway, leading to the M1 polarization of TAMs. Furthermore, macrophages with elevated Gbp2 expression significantly inhibit tumor growth in both ID8 ovarian cancer and 4T1 breast cancer models. Conversely, targeting Gbp2 diminishes the antitumor efficacy of the nanovaccine in vivo. This study offers an innovative approach to immunotherapy and elucidates a novel mechanism (Gbp2-Pin1-NFκB pathway) for remodeling TAMs.

GDP-bound Rab37 modulates M2-like tumor-associated macrophage polarization by attenuating STAT1 translocation to downregulate the type I IFN pathway.

Tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) primarily polarize into the M2-phenotype. Our previous study showed that the small GTPase Rab37 mediates IL-6 trafficking in macrophages for M2 polarization. Here, we uncover an unconventional role of Rab37, independent of vesicle trafficking, in promoting M2 polarization of TAMs. The gene profiles in wild-type and Rab37 knockout (KO) bone marrow-derived macrophages (BMDMs) were analyzed using cDNA microarray. The mechanism of Rab37 in regulating the interferon (IFN) pathway was confirmed through in vitro/vivo assays and clinical studies. Type I IFN signaling was highly enriched in BMDMs from Rab37 KO mice. Moreover, Rab37 induction and decreased type I IFN genes were observed in macrophages treated with lung cancer-conditioned medium and epigenetic drugs, indicating an epigenetic regulation of Rab37 in TAMs. Mechanistically, GDP-bound Rab37 interacted with the nuclear localization sequence of STAT1 to sequest it in the cytosol from its transcription activities, thus leading to the downregulation of IFN genes. Clinically, CD163+/Rab37+/STAT1cytosol in TAMs expression signature correlated with advanced tumor stages and poor survival of lung cancer patients. Our findings highlight the cytosolic interaction of Rab37-STAT1 in M2 TAM polarization, with CD163+/Rab37+/STAT1cytosol TAMs as a lung cancer prognosis biomarker.

MMP28 recruits M2-type tumor-associated macrophages through MAPK/JNK signaling pathway-dependent cytokine secretion to promote the malignant progression of pancreatic cancer

BackgroundCrosstalk between pancreatic cancer cells and tumor-associated macrophages (TAMs) is a critical driver of malignant progression, and plays an important role in the low response rate to immunotherapy in patients with for pancreatic cancer. Although it is known that cancer cells induce TAM infiltration and M2 polarization, the underlying mechanisms remain elusive. Herein, we identified matrix metalloproteinase 28 (MMP28), a highly expressed protein, as a key regulator of this process.MethodsImmunohistochemical staining and qRT-PCR were used to validate MMP28 as a potential marker for the prognosis of patients with pancreatic cancer. We evaluated the tumor-promoting effect of MMP28 in vitro with CCK-8, Transwell, and EdU assay and Western blotting and explored the potential mechanism of MMP28-induced M2 polarization of TAMs with a coculture system, immunofluorescence staining and flow cytometry. A subcutaneous graft tumor model was constructed to assess the tumor-promoting effect of MMP28 and its ability to induce M2 TAM infiltration.ResultsThe relevant results of this study revealed a strong correlation between MMP28 expression and TAM infiltration, with a predominance of M2-polarized TAMs in pancreatic cancer tissues. Mechanistic investigations demonstrated that MMP28 promotes the secretion of multiple cytokines, including IL-8 and VEGFA through the activation of the MAPK/JNK signaling pathway. These cytokines act as potent chemoattractants and polarizing factors for TAMs. Additionally, we discovered an interaction between MMP28 and ANXA2, which contributes to the regulation of TAM recruitment and polarization. In vivo studies confirmed the critical role of MMP28 in tumor growth and TAM infiltration. Depletion of macrophages, inhibition of JNK, or neutralization of IL-8 and VEGFA significantly suppressed tumor progression. Transcriptomic analysis suggested that IL-8 and VEGFA induce M2 TAM polarization by modulating TAM amino acid metabolism.ConclusionsCollectively, our findings elucidate a novel mechanism by which pancreatic cancer cells manipulate the tumor microenvironment through MMP28-dependent cytokine secretion, promoting TAM infiltration and M2 polarization. These results highlight MMP28 as a promising therapeutic target for pancreatic cancer.Graphical Schematic overview of the mechanisms by which MMP28 promotes the migration and polarization of TAMs. High levels of MMP28 promote the secretion of IL-8 and VEGFA by cancer cells by mediating the phosphorylation of the MAPK/JNK signalling pathway and then recruiting TAMs. IL-8 and VEGFA subsequently induce amino acid metabolism alterations in TAMs by binding to relevant receptors on TAMs, which ultimately promote the polarization of TAMs to the M2 phenotype. In addition, ANXA2 increases MMP28-mediated M2 TAM infiltration by interacting with MMP28.

Immunomodulatory role of exosome-derived content in pediatric medulloblastoma: a molecular subgroup perspective.

Medulloblastoma (MB) is the most common malignant brain tumor in children, comprising four distinct subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4. MYC amplification and metastatic dissemination are challenges in clinical management, and tumor-associated macrophages (TAMs) play an essential role in these intricate molecular processes. However, the influence of immune cells in MB metastasis and MYC-amp is unclear. Secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator facilitating communication within the tumor microenvironment, orchestrating coordinated responses among immune cells during tumor initiation, progression, and tumor dissemination. Here, we sought to elucidate the role of exosome-derived MBs in promoting specific patterns of TAM polarization across different molecular subgroups of MB cell lines. CIBERSORTx analysis using a single-cell RNA dataset revealed an increase in M0 macrophages and a decreased proportion of M2 macrophages in MB patients with tumor dissemination in the central nervous system (CNS). Cell-derived exosomes were found to secrete high levels of IL-4, IL-10, and TGF-β, indicative of a protumor M2-profile pattern. Moreover, EVs from SHH TP53-mutated, Group 3/4, and MYC-amplified MBs induced dissimilar patterns of TNF-α and/or IL-1β overexpression. This study demonstrates that exosomes from pediatric MBs promote a predominant M2-macrophage phenotype and Group 3, Group 4, SHH TP53-mutated, and MYC-amplified MBs induced a mixed M1/M2 response pattern. These findings shed light on the pivotal role of exosomes in modulating the immune response, potentially contributing to immune escape in this malignant neoplasm.

Reevaluating Calculus bovis: Modulating the liver cancer immune microenvironment via the Wnt/β-catenin pathway.

In this article, we comment on the work published by Huang et al, which explores the mechanisms by which Calculus bovis (CB) modulates the liver cancer immune microenvironment via the Wnt/β-catenin signalling pathway. The study demonstrates that active components in CB effectively inhibit the activation of the Wnt/β-catenin pathway, significantly reducing the polarization of M2 tumor-associated macrophages. Both in vivo and in vitro experiments have validated the anti-tumour effects of CB, revealing its complex mechanisms of action through the modulation of immune cell functions within the tumour microenvironment. This article highlights CB's therapeutic potential in liver cancer treatment and calls for further investigations into its mechanisms and clinical applications to develop safer, more effective options for patients. The study also revealed that key components of CB, such as bilirubin and bile acids, inhibit tumour cell proliferation and promote apoptosis through multiple pathways. Future research should explore the mechanisms of action of CB and its potential integration with existing treatments to improve the therapeutic outcomes of liver cancer patients. With multidisciplinary collaboration and advanced research, CB could become a key component of comprehensive liver cancer treatment, offering new hope for patients.

A pH-Responsive and Guanidinium-Rich Nanoadjuvant Efficiently Delivers STING Agonist for Cancer Immunotherapy.

As natural agonists of the stimulator of interferon genes (STING), cyclic dinucleotides (CDNs) have been identified as promising immunotherapies that trigger a potent immune response against tumors. However, the low stability, rapid clearance, inadequate cellular uptake, and inefficient cytosol localization heavily hinder the therapeutic efficacy of the hydrophilic and negatively charged 2', 3'-cyclic-GMP-AMP (cGAMP). How to efficiently deliver cGAMP into the endoplasmic reticulum (ER) to activate STING for immune priming remains challenging. Here, we report a pH-responsive and guanidinium-rich STING nanoagonist (nPGSA) for cytosol delivery of cGAMP. Compared with free cGAMP, nPGSA achieves up to a 37.4-fold enhancement of cellular internalization. The pH-sensitive and guanidinium-functional design facilitates quick release and endosome escape, thus enabling precise ER targeting of cGAMP and 33.9-fold amplification of STING sensibilization. Furthermore, through the modulation of tumor-associated macrophage (TAM) polarization, nPGSA elicits a potent antigen-specific cellular immune response and sustained tumor regression in melanoma- and neuroblastoma-bearing mice. Our study provides a promising strategy for the delivery of cGAMP, and it offers insights into the function of cGAMP in modulating the tumor immune microenvironment for cancer immunotherapy.

Platycodon grandiflorum-derived extracellular vesicles suppress triple-negative breast cancer growth by reversing the immunosuppressive tumor microenvironment and modulating the gut microbiota

Despite the approval of several artificial nanotherapeutics for the treatment of triple-negative breast cancer (TNBC), significant challenges, including unsatisfactory therapeutic outcomes, severe side effects, and the high cost of large-scale production, still restrict their long-term application. In contrast, plant-derived extracellular vesicles (PEVs) exhibit promising potential in cancer therapy due to their negligible systemic toxicity, high bioavailability and cost- effectiveness. In this study, we developed an alternative strategy to inhibit TNBC via Platycodon grandiflorum (PG)-derived extracellular vesicles (PGEVs). The PGEVs were isolated by ultracentrifugation and sucrose gradient centrifugation method and contained adequate functional components such as proteins, lipids, RNAs and active molecules. PGEVs exhibited remarkable stability, tolerating acidic digestion and undergoing minimal changes in simulated gastrointestinal fluid. They were efficiently taken up by tumor cells and induced increased production of reactive oxygen species (ROS), leading to tumor cell proliferation inhibition and apoptosis, particularly in the TNBC cell line 4T1. Additionally, PGEVs facilitated the polarization of tumor-associated macrophages (TAMs) toward M1 phenotype and increased the secretion of pro-inflammatory cytokines. Further in vivo investigations revealed that PGEVs efficiently accumulated in 4T1 tumors and exerted significant therapeutic effects through boosting systemic anti-tumor immune responses and modulating the gut microbiota whether administered orally or intravenously (i.v.). In conclusion, these findings highlight PGEVs as a promising natural, biocompatible and efficient nanotherapeutic candidate for treating TNBC.Graphical

Advanced Nanoprobe Strategies for Imaging Macrophage Polarization in Cancer Immunology.

Macrophages are ubiquitous within the human body and serve pivotal roles in immune surveillance, inflammation, and tissue homeostasis. Phenotypic plasticity is a hallmark of macrophages, allowing their polarization into distinct phenotypes M1 (pro-inflammatory, anti-tumor) and M2 (anti-inflammatory, pro-tumor) in response to local microenvironmental cues. In tumor tissues, the polarization of tumor-associated macrophages profoundly shapes the tumor microenvironment, influencing tumor progression, immune evasion, and metastasis. Therefore, the ability to image and monitor macrophage polarization is essential for comprehending tumor biology and optimizing therapeutic strategies. With the rapid advancement of nanomedicine, a diverse array of nanoprobes has been engineered to specifically target tumor-associated macrophages, offering new avenues for noninvasive invivo imaging and real-time monitoring of macrophage dynamics within the tumor microenvironment. This perspective highlights recent advancements in macrophage-targeting nanoprobes for imaging macrophage polarization both invitro and invivo. It also addresses the current challenges in the field, such as enhancing probe sensitivity, specificity, and biocompatibility, while outlining the future directions for the development of next-generation nanoprobes aimed at precision oncology.

Immunoglobulin-like transcript 5 polarizes M2-like tumor-associated macrophages for immunosuppression in non-small cell lung cancer.

Immune checkpoint inhibitors (ICIs) have shifted the treatment paradigm of non-small cell lung cancer (NSCLC) over the last decade. Despite notable therapeutic advancements in responders, the response rate remains limited owing to the immunosuppressive tumor microenvironment (TME). Therefore, to improve the efficacy of ICIs, it is essential to explore alternative targets or signals that mediate immunosuppression. Immunoglobulin-like transcript (ILT) 5 is a negative regulator of immune activation in myeloid cells. However, the expression and function of ILT5 in NSCLC remain unknown. Here, we found that ILT5 was highly expressed in tumor-associated macrophages (TAMs) of NSCLC tissues and predicted poor patient survival. Functionally, ILT5 induces the M2-like polarization of TAMs, which subsequently decreases the density of T cells, and increases FOXP3+T cell accumulation, leading to an immunosuppressive TME. The combination of ILT5 expression with M2-like TAM density is a more reliable biomarker of patient survival than ILT5 expression alone. ILT5 knockout mitigates the reprogramming of TAM and T cell subsets toward immunosuppressive phenotypes and inhibits tumor growth in vivo. These findings highlight that ILT5 is a potential immunotherapeutic target and a promising prognostic biomarker for NSCLC.