EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Chimeric Antigen Receptor-T Cell Therapy Decreases Distant Metastasis and Inhibits Local Recurrence Post-surgery in Mice.

Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and local recurrence of tumors, respectively. Chimeric antigen receptor (CAR)-modified T cells target residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells infusion. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.

Surgical Treatment of Osteosarcoma Induced Distant Pre-Metastatic Niche in Lung to Facilitate the Colonization of Circulating Tumor Cells.

Recently, the major challenge in treating osteosarcoma patients is the metastatic disease, most commonly in the lungs. However, the underlying mechanism of recurrence and metastasis of osteosarcoma after surgical resection of primary tumor remains unclear. This study aims to investigate whether the pulmonary metastases characteristic of osteosarcoma is associated with surgical treatment and whether surgery contributes to the formation of pre-metastatic niche in the distant lung tissue. In the current study, the authors observe the presence of circulating tumor cells in patients undergoing surgical resection of osteosarcoma which is correlated to tumor recurrence. The pulmonary infiltrations of neutrophils and Gr-1+ myeloid cells are characterized to form a pre-metastatic niche upon the exposure of circulating tumor cells after surgical resection. It is found that mitochondrial damage-associated molecular patterns released from surgical resection contribute to the formation of pre-metastatic niche in lung through IL-1ß secretion. This study reveals that surgical management for osteosarcoma, irrespective of the primary tumor, might promote the formation of postoperative pre-metastatic niche in lung which is with important implications for developing rational therapies during peri-operative period.

Immunosuppressive cells in cancer: mechanisms and potential therapeutic targets.

Immunotherapies like the adoptive transfer of gene-engineered T cells and immune checkpoint inhibitors are novel therapeutic modalities for advanced cancers. However, some patients are refractory or resistant to these therapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. Immunosuppressive cells such as myeloid-derived suppressive cells, tumor-associated macrophages, tumor-associated neutrophils, regulatory T cells (Tregs), and tumor-associated dendritic cells are critical factors correlated with immune resistance. In addition, cytokines and factors secreted by tumor cells or these immunosuppressive cells also mediate the tumor progression and immune escape of cancers. Thus, targeting these immunosuppressive cells and the related signals is the promising therapy to improve the efficacy of immunotherapies and reverse the immune resistance. However, even with certain success in preclinical studies or in some specific types of cancer, large perspectives are unknown for these immunosuppressive cells, and the related therapies have undesirable outcomes for clinical patients. In this review, we comprehensively summarized the phenotype, function, and potential therapeutic targets of these immunosuppressive cells in the tumor microenvironment.

CXCL13 as a Novel Immune Checkpoint for Regulatory B Cells and Its Role in Tumor Metastasis.

Tumor metastasis is the primary cause of mortality in patients with cancer. Several chemokines are identified as important mediators of tumor growth and/or metastasis. The level of CXCL13 has been reported to be elevated in serum or tumor tissues in patients, which mainly functions to attract B cells and follicular B helper T cells. However, the role of CXCL13 in cancer growth and metastasis is not fully explored. In the current study, we found that CXCL13 is not a strong mediator to directly promote tumor growth; however, the mice deficient in CXCL13 had far fewer pulmonary metastatic foci than did the wild-type mice in experimental pulmonary metastatic models. In addition, Cxcl13 -/- mice also had fewer IL-10-producing B cells (CD45+CD19+IL-10+) in the metastatic tumor immune microenvironment than those of wild-type C57BL/6 mice, resulting in an enhanced antitumor immunity. Notably, CXCL13 deficiency further improved the efficacy of a traditional chemotherapeutic drug (cyclophosphamide), as well as that of anti-programmed death receptor-1 immunotherapy. These results suggested that CXCL13 has an important role in regulating IL-10-producing B cells in tumor metastasis and might be a promising target for improving therapeutic efficiency and stimulating tumor immunity in future cancer therapy.

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis.

BACKGROUND: Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co-localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved. METHODS: Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography-mass spectrometry. Phosphatidylserine-containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real-time PCR, immunohistochemistry enzyme-linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies. RESULTS: The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2-like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4 (TIM4) and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression. CONCLUSIONS: This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.

Targeted and immuno-based therapies in sarcoma: mechanisms and advances in clinical trials.

Sarcomas represent a distinct group of rare malignant tumors with high heterogeneity. Limited options with clinical efficacy for the metastatic or local advanced sarcoma existed despite standard therapy. Recently, targeted therapy according to the molecular and genetic phenotype of individual sarcoma is a promising option. Among these drugs, anti-angiogenesis therapy achieved favorable efficacy in sarcomas. Inhibitors targeting cyclin-dependent kinase 4/6, poly-ADP-ribose polymerase, insulin-like growth factor-1 receptor, mTOR, NTRK, metabolisms, and epigenetic drugs are under clinical evaluation for sarcomas bearing the corresponding signals. Immunotherapy represents a promising and favorable method in advanced solid tumors. However, most sarcomas are immune "cold" tumors, with only alveolar soft part sarcoma and undifferentiated pleomorphic sarcoma respond to immune checkpoint inhibitors. Cellular therapies with TCR-engineered T cells, chimeric antigen receptor T cells, tumor infiltrating lymphocytes, and nature killer cells transfer show therapeutic potential. Identifying tumor-specific antigens and exploring immune modulation factors arguing the efficacy of these immunotherapies are the current challenges. This review focuses on the mechanisms, advances, and potential strategies of targeted and immune-based therapies in sarcomas.

Hyperprogression: A novel response pattern under immunotherapy.

Checkpoint blockade therapy has shown significant therapeutic benefits and resulted in durable responses in patients with various tumors. However, accumulating evidence has demonstrated that 4-29% of all patients with cancers with various histologies may suffer from tumor flare following such therapy. This novel tumor response pattern, termed hyperprogression, is a potentially deleterious side effect of checkpoint blockade therapy that accelerates disease progression in a subset of patients. In this review, we describe possible immune checkpoint blockade biomarkers and the epidemiology, different definitions, and predictors of hyperprogression based on the research findings and further present the available evidence supporting pathophysiological hypotheses that might explain hyperprogression during checkpoint blockade therapy. We also compare hyperprogression and pseudoprogression. Finally, we discuss areas requiring further study.

Progress in Neoantigen Targeted Cancer Immunotherapies.

Immunotherapies that harness the immune system to kill cancer cells have showed significant therapeutic efficacy in many human malignancies. A growing number of studies have highlighted the relevance of neoantigens in recognizing cancer cells by intrinsic T cells. Cancer neoantigens are a direct consequence of somatic mutations presenting on the surface of individual cancer cells. Neoantigens are fully cancer-specific and exempt from central tolerance. In addition, neoantigens are important targets for checkpoint blockade therapy. Recently, technological innovations have made neoantigen discovery possible in a variety of malignancies, thus providing an impetus to develop novel immunotherapies that selectively enhance T cell reactivity for the destruction of cancer cells while leaving normal tissues unharmed. In this review, we aim to introduce the methods of the identification of neoantigens, the mutational patterns of human cancers, related clinical trials, neoantigen burden and sensitivity to immune checkpoint blockade. Moreover, we focus on relevant challenges of targeting neoantigens for cancer treatment.

Enhancement of the antitumor effect of HER2-directed CAR-T cells through blocking epithelial-mesenchymal transition in tumor cells.

The efficacy of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors is far from satisfactory. In this study, we investigated the influence of epithelial-mesenchymal transition (EMT) on the antitumor effect of CAR-T cells and explored the potential efficacy of combining CAR-T cells with inhibitors targeting EMT. We successfully induced EMT in tumor cells with TGF-ß1, and the antitumor effect of HER2-directed CAR-T cells was significantly suppressed by EMT. Upregulation of PD-L1 was observed in tumor cells undergoing EMT, and change in PD-L1 expression during the EMT process was dependent on the MEK/ERK and PI3K/Akt pathways. Inhibition of the TGF-ß1 pathway could block the EMT process in tumor cells and restore their susceptibility to HER2-directed CAR-T cells in vitro. In addition, targeting the TGF-ß1 pathway significantly enhanced the antitumor effect of HER2-directed CAR-T cells in vivo. Our findings suggest that blocking EMT could potently enhance the antitumor effect of CAR-T cells, which provides a promising approach to improving the therapeutic efficacy of CAR-T cell therapy in solid tumors.

IL-18R-dependent and independent pathways account for IL-18-enhanced antitumor ability of CAR-T cells.

Interleukin-18 (IL-18) has been demonstrated to augment the antitumor capacity of chimeric antigen receptor-T cells (CAR-T) but the underlying mechanisms are largely unknown. Here we explored the effects and mechanisms of exogenous IL-18 on the antitumor response of CAR-T cells. IL-18 boosted the cytotoxicity of human epidermal growth factor receptor-2 (HER2)-specific CAR-T cells ex vivo and enhanced the antitumor efficacy of the CAR-T cells in immunodeficient mice, moreover, IL-18 improved the antitumor capacity of OVA-specific T cells in immunocompetent mice, indicating the universal enhancing function of IL-18 for adoptive cell therapy. To address the roles of IL-18 receptor (IL-18R) in the enhancing function, we evaluated the effects of IL-18R knockout (IL-18R-/-) condition in immunocompetent host and CAR-T cells on the IL-18-enhanced antitumor activities. Interestingly, IL-18 persisted to improve the antitumor ability of IL-18R intact CAR-T cells in IL-18R-/- mice. For IL-18R-/- CAR-T cells, however, IL-18 still holds the enhancing ability to boost the antitumor efficacy in IL-18R-/- mice, albeit the ex vivo tumor-killing ability was lower than that of IL-18R intact CAR-T cells, indicating that IL-18R-independent pathway is involved in the enhancement. Furthermore, tagged IL-18 binded to the membrane of IL-18R-/- splenic and lymph node cells and IL-18R intact and IL-18R-/- CAR-T cells showed distinct transcriptomic profiles when stimulated by IL-18. These data demonstrate that IL-18R-independent pathways contribute to functions of IL-18.

The approved gene therapy drugs worldwide: from 1998 to 2019.

With the improvement of gene vectors, the rise of chimeric antigen receptor T cell immunotherapy and breakthroughs in the genome editing technology, gene therapy had once again returned to the central stage of disease treatment. It had brought new choices to clinical therapy of diseases such as tumors and genetic diseases, and had changed the status quo of treatment for monogenic disorders and diffuse large B-cell lymphoma. Until August 2019, 22 gene medicines had been approved by the drug regulatory agencies from various countries, but there were few relevant reviews of combing these drugs systematically. Consequently, this review summarizes the gene therapy drugs approved worldwide from 1998 to 2019 in details, including names, indications, dates of approval, companies, vectors, the applied technologies and mechanisms of gene therapy drugs, etc. Furthermore, the gene therapy drugs were classified and addressed in accordance with the employed vectors. Gene therapy had gradually been accepted by the government and the public since 1980s, and have become a new and important alternative to existing treatments for human diseases in the past few years. Therefore, gene therapy drugs, with safe vectors and advanced biotechnologies, would play a greater role in the prevention and treatment of human diseases in future.

Correction: Inhibition of Stat3 signaling pathway by nifuroxazide improves antitumor immunity and impairs colorectal carcinoma metastasis.

Since publication of this article, the authors have noticed that there were errors in Fig. 1b (the CT 26 cells colony formation images) and Fig. 7c (the vehicle group images). As a result of the misfiling of the data during preparation of figures, incorrect images were inadvertently inserted in these figures.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RNA-Seq profiling of circular RNA in human lung adenocarcinoma and squamous cell carcinoma.

Emerging evidences demonstrate that circular RNAs (circRNAs) are abnormally expressed in tumors and could serve as prognostic markers for cancers. However, the expression patterns and clinical implications of circRNAs in non-small cell lung cancer (NSCLC) remain obscure. In this study, we profiled circRNA expressions in 10 pairs of lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) after ribosomal RNA-depletion and RNase R digestion to enrich circRNAs. Combining five circRNA computational programs, we found that LUAD and LUSC not only share common expression patterns, but also exhibit distinct circRNA expression signatures. Moreover, the Receiver Operating Characteristic (ROC) curve analysis indicated that hsa_circ_0077837 and hsa_circ_0001821 could serve as potential biomarkers for both LUAD and LUSC, while hsa_circ_0001073 and hsa_circ_0001495 could be diagnostic/subtyping marker for LUAD and LUSC, respectively. Therefore, our findings highlight the important diagnostic potential of circRNAs in NSCLC.

MicroRNA Biogenesis is Enhanced by Liposome-Encapsulated Pin1 Inhibitor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is in an urgent need of new, effective therapies to reduce morbidity and mortality. We have previously demonstrated that peptidyl-prolyl cis/trans isomerase Pin1 is a potential target for HCC therapy, due to its pivotal role in HCC development through regulating miRNA biogenesis, and discovered the small molecule API-1 as a novel and specific Pin1 inhibitor. Despite its significant anti-HCC activity, the low water solubility and in vivo bioavailability of API-1 limit its clinical application. To address these issues, we herein developed a liposomal formulation of API-1 to improve API-1 delivery and enhance its anti-HCC efficacy. Methods: We designed and developed a nanoscale liposomal formulation of API-1, named as API-LP. Subsequently, the mean diameter, polydispersity, zeta potential, encapsulation efficiency and thermal properties of the optimization API-LP were characterized. The enhanced anti-HCC activity and the molecular mechanism of API-LP were investigated both in vitro and in vivo. Finally, the safety and pharmacokinetic property of API-LP were evaluated systematically. Results: API-LP had good formulation characteristics and exhibited an enhanced in vitro activity of suppressing proliferation and migration of HCC cells when compared with free API-1. The mechanism study showed that API-LP upregulated miRNA biogenesis via inhibiting Pin1 activity followed by restoring the nucleus-to-cytoplasm export of XPO5. Because of the increased delivery efficiency, API-LP displayed a stronger ability to promote miRNA biogenesis than free API-1. Importantly, API-LP displayed higher systemic exposure than free API-1 in mice without apparent toxicity, resulting in an enhanced tumor inhibition in xenograft mice. Conclusion: The development and assessment of API-LP provide an attractive and safe anti-HCC agent, highlighting the miRNA-based treatment for human cancers.

Circular RNA F-circSR derived from SLC34A2-ROS1 fusion gene promotes cell migration in non-small cell lung cancer.

Cancer-associated chromosomal translocations are reported to generate oncogenic circular RNA (circRNA), contributing to tumorigenesis. The fusion gene SLC34A2-ROS1 (solute carrier family 34 member 2 and ROS proto-oncogene 1) plays an important role in non-small cell lung cancer (NSCLC) progression. However, whether SLC34A2-ROS1 gene can produce circRNA remains unknown. Here, we identified two novel circRNAs (F-circSR1 and F-circSR2) generated from SLC34A2-ROS1 fusion gene, while F-circSR1 has higher expression than F-circSR2. Functional studies through gain- and loss-of-function strategies showed that both F-circSRs promote cell migration in lung cancer cells, whereas they have little effect on cell proliferation. Using the minigene GFP reporter assay, we verified that the flanking complementary sequences with canonical splicing sites are essential for F-circSR biogenesis. Therefore, our findings demonstrate the oncogenic role of F-circSR in NSCLC and highlight its therapeutic potential.

Novel ROR1 inhibitor ARI-1 suppresses the development of non-small cell lung cancer.

Limited drug response and severe drug resistance confer the high mortality of non-small-cell lung cancer (NSCLC), a leading cause of cancer death worldwide. There is an urgent need for novel treatment against NSCLC. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is aberrantly overexpressed and participats in NSCLC development and EGFR-TKIs-induced drug resistance. Increasing evidences indicate that oncogenic ROR1 is a potential target for NSCLC therapy. However, nearly no ROR1 inhibitor was reported until now. Here, combining the computer-aided drug design and cell-based activity screening, we discover (R)-5,7-bis(methoxymethoxy)-2-(4-methoxyphenyl)chroman-4-one (ARI-1) as a novel ROR1 inhibitor. Biological evaluation demonstrates that ARI-1 specifically targets the extracellular frizzled domain of ROR1 and potently suppresses NSCLC cell proliferation and migration by regulating PI3K/AKT/mTOR signaling in a ROR1-dependent manner. Moreover, ARI-1 significantly inhibits tumor growth in vivo without obvious toxicity. Intriguingly, ARI-1 is effective to EGFR-TKIs-resistant NSCLC cells with high ROR1 expression. Therefore, our work suggests that the ROR1 inhibitor ARI-1 is a novel drug candidate for NSCLC treatment, especially for EGFR-TKIs-resisted NSCLC with high ROR1 expression.

Exosomal tRNA-derived small RNA as a promising biomarker for cancer diagnosis.

tRNA-derived small RNA (tsRNA) is a novel regulatory small non-coding RNA and participates in diverse physiological and pathological processes. However, the presence of tsRNAs in exosome and their diagnostic potential remain unclear. In this study, we took advantage of small RNA-seq technology to profile exosomal tsRNAs from cell culture medium and plasma, and found ubiquitous presence of tsRNAs in exosome. To explore the potential value of tsRNA for cancer diagnosis, we compared exosomal tsRNA levels between liver cancer patients and healthy donors, revealing that tsRNAs were dramatically increased in plasma exosomes of liver cancer patients. Importantly, patients with liver cancer exhibited significantly higher levels of four tsRNAs (tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5 and tRNA-GluCTC-5) in plasma exosome, demonstrating that plasma exosomal tsRNA could serve as a novel diagnostic biomarker. Taken together, our results not only expand non-coding RNA species in exosome, but also highlight the potential of tsRNAs as a promising biomarker for cancer diagnosis.

Potential roles and targeted therapy of the CXCLs/CXCR2 axis in cancer and inflammatory diseases.

The chemokine receptor CXCR2 and its ligands are implicated in the progression of tumours and various inflammatory diseases. Activation of the CXCLs/CXCR2 axis activates multiple signalling pathways, including the PI3K, p38/ERK, and JAK pathways, and regulates cell survival and migration. The CXCLs/CXCR2 axis plays a vital role in the tumour microenvironment and in recruiting neutrophils to inflammatory sites. Extensive infiltration of neutrophils during chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases. Chronic inflammation is considered to be closely correlated with initiation of cancer. In addition, immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) against T cells attenuate the anti-tumour effects of T cells and promote tumour invasion and metastasis. Over the last several decades, many therapeutic strategies targeting CXCR2 have shown promising results and entered clinical trials. In this review, we focus on the features and functions of the CXCLs/CXCR2 axis and highlight its role in cancer and inflammatory diseases. We also discuss its potential use in targeted therapies.

Preclinical Evaluation of Chimeric Antigen Receptor-Modified T Cells Specific to Epithelial Cell Adhesion Molecule for Treating Colorectal Cancer.

Chimeric antigen receptor-modified T cells (CAR-T cells) have emerged as a promising cancer immunotherapy for solid tumors. Epithelial cell adhesion molecule (EpCAM) is overexpressed in a variety of tumors and is recognized as a biomarker for circulating tumor cells and cancer stem cells, representing an attractive target for adoptive T-cell immunotherapy. This study generated third-generation CAR-T cells with redirected specificity to EpCAM (EpCAM CAR-T) by lentiviral vector. The study demonstrated that EpCAM CAR-T cells can elicit lytic cytotoxicity to target cells in an EpCAM-dependent manner and secrete cytotoxic cytokines, including interferon gamma and tumor necrosis factor alpha. Furthermore, adoptive transfer of EpCAM CAR-T cells significantly delayed tumor growth and formation in xenograft models. In addition, the safety evaluation showed that CAR-T cells have no systemic toxicity in mice. The data confirmed the antitumor ability and safety of CAR-T cells targeting EpCAM and may provide a new target for CAR-T cell therapies in treating solid tumors.