Direct identification of T cell epitopes in cancer tissues.

Prediction of tumor-specific T cell epitopes is an important part of cancer immunotherapies. In the past, tumor-specific T cell epitopes were identified by mapping the epitopes on the known cancer-testis antigens and tumor-associated antigens or antigens that react to the T cells induced by the cancer vaccine therapy. More recently, in silico prediction of mutation-associated neoepitopes from the whole-exome sequencing (WES) results has become another approach. However, although this approach often identifies many predicted peptides, only few have been shown to be immunogenic. Mass spectrometry (MS) has also been used to directly identify the T cell epitopes presented on tumor cell by eluting the peptides from human leukocyte antigens (HLA) class I and class II molecules. This approach of identifying neoepitopes was demonstrated to be feasible in high tumor mutation burden (TMB) tumors such as melanoma. However, identifying low-TMB-tumor-specific T cell epitopes has been challenging. Recently, Fujiwara et al. reported their successful result in identifying T cell epitopes in a low TMB tumor, namely pancreatic ductal adenocarcinoma (PDAC). Using the MS approach, they identified T cell epitopes shared by multiple pancreatic cancer patients with different HLA types. Moreover, they demonstrated that the identified epitopes bound non-matched HLA molecules and induced T cell response in peripheral T cells from non-HLA-type matched patients. Their study has opened a new venue for identifying T cell epitopes in a non-immunogenic tumor such as PDAC for the design and development of vaccine and T cell therapy.

α-SMA+ cancer-associated fibroblasts increased tumor enhancement ratio on contrast-enhanced multidetector-row computed tomography in stages I-III colon cancer.

BACKGROUND AND AIM: Our prior research revealed that the tumor enhancement ratio (TER) on triphasic abdominal contrast-enhanced MDCT (CE-MDCT) scans was a prognostic factor for patients with stages I-III colon cancer. Building upon this finding, the present study aims to investigate the proteomic changes in colon cancer patients with varying TER values. METHODS: TER was analyzed on preoperative triphasic CE-MDCT scans of 160 stages I-III colon cancer patients. The survival outcomes of those in the low-TER and high-TER groups were compared. Proteomic analysis on colon cancer tissues was performed by mass spectrometry (MS) and verified by immune-histological chemistry (IHC) assays. In vivo, mouse xenograft models were employed to test the function of target proteins identified through the MS. CE-MDCT scans were conducted on mice xenografts, and the TER values were compared. RESULTS: Patients in the high-TER group had a significantly worse prognosis than those in the low-TER group. Proteomic analysis of colon cancer tissues revealed 153 differentially expressed proteins between the two groups. A correlation between TER and the abundance of α-SMA protein in tumor tissue was observed. IHC assays further confirmed that α-SMA protein expression was significantly increased in high-TER colon cancer, predominantly in cancer-associated fibroblasts (CAFs) within the cancer stroma. Moreover, CAFs promoted the growth of CRC xenografts in vivo and increased TER. CONCLUSIONS: Our study identified the distinct protein changes in colon cancer with low and high TER for the first time. The presence of CAFs may promote the growth of colon cancer and contribute to an increased TER.

CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer.

BACKGROUND & AIMS: BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). METHODS: We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. RESULTS: AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. CONCLUSIONS: Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Molecular characterization of colorectal mucinous adenocarcinoma and adenocarcinoma, not otherwise specified, identified by multiomic data analysis.

Adenocarcinoma not otherwise specified (AC) and mucinous adenocarcinoma (MC) have different biological behaviors and clinical features. We utilized our previous proteomic data and public transcriptome, single-cell transcriptome, and spatial transcriptome databases to profile the molecular atlas of the tumor microenvironments of MC, AC, and normal colon tissues. By exploring the general and specific molecular features of AC and MC, we found that AC was immune-active but exposed to a hypoxic microenvironment. MC cells could protect against DNA damage, and the microenvironment was unfavorable to leukocyte transendothelial migration. We identified several potential molecular and cellular targets of AC and MC for future research. We also highlighted that the major difference between AC and MC was not the variety of cell types and functions but possibly cell interactions. Stromal and epithelial cell interactions play important roles in both MC and AC, but different regulatory pathways were involved.

FOSL2 promotes intertumoral infiltration of T cells and increases pathological complete response rates in locally advanced rectal cancer patients.

The outcome of neoadjuvant chemoradiotherapy (nCRT) remains highly unpredictable for individuals with locally advanced rectal cancer (LARC). We set out to characterize effective biomarkers that promote a pathological complete response (pCR). We quantified the abundances of 6483 high-confidence proteins in pre-nCRT biopsies of 58 LARC patients from two hospitals with pressure cycling technology (PCT)-assisted pulse data-independent acquisition (PulseDIA) mass spectrometry. Compared with non-pCR patients, pCR patients achieved long-term disease-free survival (DFS) and had higher tumor immune infiltration, especially CD8+ T cell infiltration, before nCRT. FOSL2 was selected as the candidate biomarker for predicting pCR and was found to be significantly upregulated in pCR patients, which was verified in another 54 pre-nCRT biopsies of LARC patients by immunohistochemistry. FOSL2 expression was able to predict pCR by multiple reaction monitoring (MRM) with high efficiency (Area under curve (AUC) = 0.939, specificity = 1.000, sensitivity = 0.850), and high FOSL2 expression was associated with long-term DFS (p = 0.044). When treated with simulated nCRT, FOSL2 sufficiency resulted in more significant inhibition of cell proliferation, and more significant promotion of cell cycle arrest and cell apoptosis. Moreover, CXCL10 secretion with abnormal cytosolic dsDNA accumulation was found in FOSL2-wildtype (FOSL2-WT) tumor cells over nCRT, which might elevate CD8+ T-cell infiltration and CD8+ T-cell-mediated cytotoxicity to promote nCRT-induced antitumor immunity. Our study revealed proteomic profiles in LARC patients before nCRT and highlighted immune activation in the tumors of patients who achieved pCR. We identified FOSL2 as a promising biomarker to predict pCR and promote long-term DFS by contributing to CD8+ T-cell infiltration.

Direct identification of HLA class I and class II-restricted T cell epitopes in pancreatic cancer tissues by mass spectrometry.

BACKGROUND: Identifying T cell epitopes on pancreatic ductal adenocarcinoma (PDAC) associated antigens or neoantigens has been a challenge. In this study, we attempted to identify PDAC T cell epitopes by mass spectrometry (MS). METHODS: We isolated HLA class I (HLA-I) and HLA class II (HLA-II)-restricted peptides, respectively, from tissues of human PDAC by using the pan-HLA-I or pan-HLA-II affinity purification column and identified T cell epitopes by peptidome analysis with MS. RESULTS: Through peptidome analysis, we identified T cell epitopes shared by multiple patients with different HLA types and those containing sequences of both anti-HLA-I and HLA-II antibodies-affinity purified peptides. The identified epitopes bound non-matched HLA molecules and induced T cell response in peripheral T cells from both HLA-type matched and non-matched patients. Peptides containing both HLA class I and class II epitopes were able to induce polyfunctional cytokine responses in peripheral T cells. CONCLUSIONS: T cell epitopes in PDAC can be discovered by the MS approach and can be designed into vaccine and TCR-T cell therapies for both HLA-type matched and non-matched patients.

Artificial intelligence defines protein-based classification of thyroid nodules.

Determination of malignancy in thyroid nodules remains a major diagnostic challenge. Here we report the feasibility and clinical utility of developing an AI-defined protein-based biomarker panel for diagnostic classification of thyroid nodules: based initially on formalin-fixed paraffin-embedded (FFPE), and further refined for fine-needle aspiration (FNA) tissue specimens of minute amounts which pose technical challenges for other methods. We first developed a neural network model of 19 protein biomarkers based on the proteomes of 1724 FFPE thyroid tissue samples from a retrospective cohort. This classifier achieved over 91% accuracy in the discovery set for classifying malignant thyroid nodules. The classifier was externally validated by blinded analyses in a retrospective cohort of 288 nodules (89% accuracy; FFPE) and a prospective cohort of 294 FNA biopsies (85% accuracy) from twelve independent clinical centers. This study shows that integrating high-throughput proteomics and AI technology in multi-center retrospective and prospective clinical cohorts facilitates precise disease diagnosis which is otherwise difficult to achieve by other methods.

Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy.

BACKGROUND & AIMS: The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. METHODS: A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. RESULTS: The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. CONCLUSIONS: This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

Immune cell atlas of cholangiocarcinomas reveals distinct tumor microenvironments and associated prognoses.

BACKGROUND: Immunotherapy has demonstrated a limited clinical efficacy in approximately 5% of cholangiocarcinoma. The main challenges for an effective immunotherapy response in cholangiocarcinoma arise from the tumor microenvironment, which is poorly understood. METHODS: For a comprehensive analysis of the tumor microenvironment in cholangiocarcinoma, we performed multiplex immunohistochemistry with two 15-marker immune panels and Nanostring assays for a comprehensive analysis of 104 surgically resected cholangiocarcinomas including intrahepatic, hilar, and distal cholangiocarcinoma. We also validated some key findings with a batch integration analysis of published single cell RNA sequencing data. RESULTS: This study found that natural killer cells occupy the largest immune cell compartment in cholangiocarcinoma. Granzyme-B+CD8+ effector T cells are significantly associated with better overall survival in both intrahepatic and distal cholangiocarcinoma. Above 85% of intrahepatic cholangiocarcinomas with higher density of PD-1-EOMES-CD8+ effector T cells are associated with long-term survival. However, only the density of PD-1-EOMES-CD8+ T cells in the tumor areas, but not in the peripheries of the tumors, is prognostic. In all three cholangiocarcinoma subtypes, T regulator cells are significantly associated with a poor prognosis; however, M1 and M2 tumor-associated macrophages or PD-L1+ tumor-associated macrophage demonstrate different prognostic values. Combining PD-L1+ M1 or M2, PD-L1- M1 or M2 tumor-associated macrophages, and T regulator cells to subgroup intrahepatic and distal cholangiocarcinoma, the prognosis is significantly better distinguished. Moreover, PD-L1- M2 tumor-associated macrophages is associated with a good prognosis in intrahepatic and distal cholangiocarcinoma, suggesting this subtype of M2 tumor-associated macrophages may be antitumoral. Interestingly, lower densities of various types of immunosuppressive cells are associated with decreased infiltration of effector T cells in distal and hilar cholangiocarcinoma, but not in intrahepatic cholangiocarcinoma. In intrahepatic cholangiocarcinoma, PD-L1+ tumor-associated macrophages exert their immunosuppressive function likely through promoting T cell exhaustion. CONCLUSIONS: This study suggests that the densities of Granzyme-B+CD8+ effector T cells and non-exhausted PD-1-EOMES-CD8+ T cells and the PD-L1 status in the tumor-associated macrophages are prognostic makers in cholangiocarcinomas. The study also supports targeting PD-L1+ tumor-associated macrophages as the immunotherapy for cholangiocarcinoma.

HomeoboxC6 promotes metastasis by orchestrating the DKK1/Wnt/ß-catenin axis in right-sided colon cancer.

Patients with right-sided colon cancer (RCC) generally have a poorer prognosis than those with left-sided colon cancer (LCC). We previously found that homeobox C6 (HOXC6) was the most significantly upregulated gene in RCC compared to LCC. However, it remains unclear whether HOXC6 plays a role in tumor proliferation and metastasis. Our study aimed to explore the potential oncogenic role and the detailed molecular mechanism of HOXC6 in RCC. In this study, HOXC6 was validated to be overexpressed in RCC and associated with poor prognosis. Furthermore, overexpression of HOXC6 promoted the migration and invasion of colon cancer cells through inducing EMT by activating the Wnt/ß-catenin signaling pathway and inhibition of DKK1 secretion. Lastly, we preliminary explored the translational effect of HOXC6 and found that silencing of HOXC6 made HCT116 and HT29 cells more sensitive to irinotecan.

DPHL: A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

Application of exosomes as liquid biopsy in clinical diagnosis.

Liquid biopsy refers to the sampling and molecular analysis of the biofluids of circulating tumor cells, extracellular vesicles, nucleic acids, and so forth. Exosomes are small extracellular vesicles with sizes between 30-150 nm. They are secreted by multivesicular bodies through exocytosis in live cells and can participate in intercellular communication due to their contents, including nucleic acids, proteins, and lipids. Herein, we investigate publication frequencies on exosomes over the past 10 years, and review recent clinical studies on liquid biopsy of exosomes in the fields of oncology, pregnancy disorders, cardiovascular diseases, and organ transplantation. We also describe the advantages of exosomes as an effective liquid biopsy tool and the progression of exosome extraction methods. Finally, we depict the commercial development of exosome research and discuss the future role of exosomes in liquid biopsy.

A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry.

Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. Abbreviations: FGA: fibrinogen α chain; CRC: colorectal cancer; crEVs: circulating extracellular vesicles; EV: extracellular vesicles;MS: mass spectrometry; WB: Western blotting; ROC: receiver operating characteristic; PRM: Parallel Reaction Monitoring; GPC1: Glypican-1; GO: Gene ontology; TEM: transmission electron microscopy; FN1: Fibronectin 1; HP: haptoglobin; TMT: Tandem Mass Tag; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; DIA: data-independent acquisition; DDA: data-dependent acquisition; CiRT: Common internal Retention Time standards;AGC: Automatic gain control; AUC: area under curve.

SP1-induced lncRNA TINCR overexpression contributes to colorectal cancer progression by sponging miR-7-5p.

Mounting evidences have indicated that long noncoding RNAs (lncRNAs) play pivotal roles in human diseases, especially in cancers. Recently, TINCR was proposed to be involved in tumor progression. However, its role in colorectal cancer (CRC) remains elusive. In our study, we found that SP1-induced TINCR was significantly upregulated in CRC tissues and cell lines. Moreover, cox multivariate survival analysis revealed that high TINCR was an independent predictor of poor overall survival (OS). Functionally, knockdown of TINCR obviously suppressed CRC cells proliferation, migration and invasion in vitro, and inhibited CRC cells growth and metastasis in vivo. Mechanistically, we identified TINCR could act as a miR-7-5p sponge using RNA pull down, luciferase reporter and RIP assays. Furthermore, we showed that TINCR might promote CRC progression via miR-7-5p-mediated PI3K/Akt/mTOR signaling pathway. Lastly, we revealed that plasma TINCR expression was upregulated in CRC when compared to healthy controls and could be a promising diagnostic biomarker for CRC. Based on above results, our data indicated that TINCR might serve as a potential diagnostic and prognostic biomarker for CRC.

The inflammatory cytokine IL-6 induces FRA1 deacetylation promoting colorectal cancer stem-like properties.

Colorectal cancer (CRC) has long been known for its tight association with chronic inflammation, thought to play a key role in tumor onset and malignant progression through the modulation of cancer stemness. However, the underlying molecular and cellular mechanisms are still largely elusive. Here we show that the IL-6/STAT3 inflammatory signaling axis induces the deacetylation of FRA1 at the Lys-116 residue located within its DNA-binding domain. The HDAC6 deacetylase underlies this key modification leading to the increase of FRA1 transcriptional activity, the subsequent transactivation of NANOG expression, and the acquisition of stem-like cellular features. As validated in a large (n = 123) CRC cohort, IL-6 secretion was invariably accompanied by increased FRA1 deacetylation at K116 and an overall increase in its protein levels, coincident with malignant progression and poor prognosis. Of note, combined treatment with the conventional cytotoxic drug 5-FU together with Tubastatin A, a HDAC6-specific inhibitor, resulted in a significant in vivo synergistic inhibitory effect on tumor growth through suppression of CRC stemness. Our results reveal a novel transcriptional and posttranslational regulatory cross-talk between inflammation and stemness signaling pathways that underlie self-renewal and maintenance of CRC stem cells and promote their malignant behavior. Combinatorial treatment aimed at the core regulatory mechanisms downstream of IL-6 may offer a novel promising approach for CRC treatment.

Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis.

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

Exosomes derived from pancreatic cancer cells induce insulin resistance in C2C12 myotube cells through the PI3K/Akt/FoxO1 pathway.

Prospective epidemiological studies have consistently suggested that pancreatic cancer-associated new-onset diabetes mellitus (PC-DM) represents a potential platform for early diagnose of pancreatic cancer (PC). Despite the studies performed, the mechanism behind this phenomenon remains ambiguous. In this study, we explored the effects of two types of exosomes released by murine pancreatic cancer and ductal epithelial cells on murine skeletal muscle cells. The results show that PC-derived exosomes can readily enter C2C12 myotubes, triggering lipidosis and glucose intake inhibition. We also demonstrate that PC-derived exosomes can inhibit insulin and PI3K/Akt signalling, in which insulin-induced FoxO1 nuclear exclusion is preserved and Glut4 trafficking is impaired. Microarray and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses show that exosomal microRNAs (miRNAs) probably play key roles in this process, an assumption that is corroborated by in vitro studies. These results confirm that the insulin resistance (IR) of skeletal muscle cells is governed by PC-derived exosomes through the insulin and PI3K/Akt/FoxO1 signalling pathways, where exosomal miRNAs potentially contribute to this phenomenon. These novel findings pave the way towards a comprehensive understanding of the cancer theories: "metabolic reprogramming" and "metabolic crosstalk".

The functions and clinical applications of tumor-derived exosomes.

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm. They can be secreted by all cell types and transfer information in the form of their contents, which include proteins, lipids and nucleic acids, to other cells throughout the body. They have roles in normal physiological processes as well as in disease development. Here, we review recent findings regarding tumor-derived exosomes, including methods for their extraction and preservation. We also describe the actions of exosomes in tumorigenesis. The exosomal antigen-presenting effect during antitumor immune responses and its suppressive function in immune tolerance are discussed. Finally, we describe the potential application of exosomes to cancer therapy and liquid biopsy.