Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

Comparative RNA-Seq Analysis Revealed Tissue-Specific Splicing Variations during the Generation of the PDX Model.

Tissue-specific gene expression generates fundamental differences in the function of each tissue and affects the characteristics of the tumors that are created as a result. However, it is unclear how much the tissue specificity is conserved during grafting of the primary tumor into an immune-compromised mouse model. Here, we performed a comparative RNA-seq analysis of four different primary-patient derived xenograft (PDX) tumors. The analysis revealed a conserved RNA biotype distribution of primary-PDX pairs, as revealed by previous works. Interestingly, we detected significant changes in the splicing pattern of PDX, which was mainly comprised of skipped exons. This was confirmed by splicing variant-specific RT-PCR analysis. On the other hand, the correlation analysis for the tissue-specific genes indicated overall strong positive correlations between the primary and PDX tumor pairs, with the exception of gastric cancer cases, which showed an inverse correlation. These data propose a tissue-specific change in splicing events during PDX formation as a variable factor that affects primary-PDX integrity.

RNA Editing Enzyme ADAR1 Suppresses the Mobility of Cancer Cells via ARPIN.

Deamination of adenine or cytosine in RNA, called RNA editing, is a constitutively active and common modification. The primary role of RNA editing is tagging RNA right after its synthesis so that the endogenous RNA is recognized as self and distinguished from exogenous RNA, such as viral RNA. In addition to this primary function, the direct or indirect effects on gene expression can be utilized in cancer where a high level of RNA editing activity persists. This report identified actin-related protein 2/3 complex inhibitor (ARPIN) as a target of ADAR1 in breast cancer cells. Our comparative RNA sequencing analysis in MCF7 cells revealed that the expression of ARPIN was decreased upon ADAR1 depletion with altered editing on its 3'UTR. However, the expression changes of ARPIN were not dependent on 3'UTR editing but relied on three microRNAs acting on ARPIN. As a result, we found that the migration and invasion of cancer cells were profoundly increased by ADAR1 depletion, and this cellular phenotype was reversed by the exogenous ARPIN expression. Altogether, our data suggest that ADAR1 suppresses breast cancer cell mobility via the upregulation of ARPIN.

MHC II immunogenicity shapes the neoepitope landscape in human tumors.

Despite advances in predicting physical peptide-major histocompatibility complex I (pMHC I) binding, it remains challenging to identify functionally immunogenic neoepitopes, especially for MHC II. By using the results of >36,000 immunogenicity assay, we developed a method to identify pMHC whose structural alignment facilitates T cell reaction. Our method predicted neoepitopes for MHC II and MHC I that were responsive to checkpoint blockade when applied to >1,200 samples of various tumor types. To investigate selection by spontaneous immunity at the single epitope level, we analyzed the frequency spectrum of >25 million mutations in >9,000 treatment-naive tumors with >100 immune phenotypes. MHC II immunogenicity specifically lowered variant frequencies in tumors under high immune pressure, particularly with high TCR clonality and MHC II expression. A similar trend was shown for MHC I neoepitopes, but only in particular tissue types. In summary, we report immune selection imposed by MHC II-restricted natural or therapeutic T cell reactivity.

The role of miRNAs in T helper cell development, activation, fate decisions and tumor immunity.

T helper (Th) cells are central members of adaptive immunity and comprise the last line of defense against pathogen infection and malignant cell invasion by secreting specific cytokines. These cytokines then attract or induce the activation and differentiation of other immune cells, including antibody-producing B cells and cytotoxic CD8+ T cells. Therefore, the bidirectional communication between Th cells and tumor cells and their positioning within the tumor microenvironment (TME), especially the tumor immune microenvironment (TIME), sculpt the tumor immune landscape, which affects disease initiation and progression. The type, number, and condition of Th cells in the TME and TIME strongly affect tumor immunity, which is precisely regulated by key effectors, such as granzymes, perforins, cytokines, and chemokines. Moreover, microRNAs (miRNAs) have emerged as important regulators of Th cells. In this review, we discuss the role of miRNAs in regulating Th cell mediated adaptive immunity, focusing on the development, activation, fate decisions, and tumor immunity.

Prediction the clinical EPR effect of nanoparticles in patient-derived xenograft models.

Many preclinically tested nanoparticles in existing animal models fail to be directly translated into clinical applications because of their poor resemblance to human cancer. Herein, the enhanced permeation and retention (EPR) effect of glycol chitosan nanoparticles (CNPs) in different tumor microenvironments (TMEs) was compared using different pancreatic tumor models, including pancreatic cancer cell line (BxPC3), patient-derived cancer cell (PDC), and patient-derived xenograft (PDX) models. CNPs were intravenously injected into different tumor models, and their accumulation efficiency was evaluated using non-invasive near-infrared fluorescence (NIRF) imaging. In particular, differences in angiogenic vessel density, collagen matrix, and hyaluronic acid content in tumor tissues of the BxPC3, PDC, and PDX models greatly affected the tumor-targeting efficiency of CNPs. In addition, different PDX models were established using different tumor tissues of patients to predict the clinical EPR effect of CNPs in inter-patient TMEs, wherein the gene expression levels of PECAM1, COL4A1, and HAS1 in human tumor tissues were observed to be closely related to the EPR effect of CNPs in PDX models. The results suggested that the PDX models could mimic inter-patient TMEs with different blood vessel structures and extracellular matrix (ECM) content that critically affect the tumor-targeting ability of CNPs in different pancreatic PDX models. This study provides a better understanding of the heterogeneity and complexity of inter-patient TMEs that can predict the response of various nanoparticles in individual tumors for personalized cancer therapy.

Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis.

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

Targeting TCTP sensitizes tumor to T cell-mediated therapy by reversing immune-refractory phenotypes.

Immunotherapy has emerged as a powerful approach to cancer treatment. However, immunotherapeutic resistance limits its clinical application. Therefore, identifying immune-resistant factors, which can be targeted by clinically available drugs and it also can be a companion diagnostic marker, is needed to develop combination strategies. Here, using the transcriptome data of patients, and immune-refractory tumor models, we identify TCTP as an immune-resistance factor that correlates with clinical outcome of anti-PD-L1 therapy and confers immune-refractory phenotypes, decreased T cell trafficking to the tumor and resistance to cytotoxic T lymphocyte-mediated tumor cell killing. Mechanistically, TCTP activates the EGFR-AKT-MCL-1/CXCL10 pathway by phosphorylation-dependent interaction with Na, K ATPase. Furthermore, treatment with dihydroartenimsinin, the most effective agent impending the TCTP-mediated-refractoriness, synergizes with T cell-mediated therapy to control immune-refractory tumors. Thus, our findings suggest a role of TCTP in promoting immune-refractoriness, thereby encouraging a rationale for combination therapies to enhance the efficacy of T cell-mediated therapy.

Exosome as a Delivery Vehicle for Cancer Therapy.

Exosomes are small extracellular vesicles that are naturally produced and carry biomolecules such as proteins, microRNAs, and metabolites. Because of their small size and low level of biomolecule expression, the biological function of exosomes has only been identified recently. Despite the short history of investigation, exosomes seem to have remarkable potential as a delivery vehicle. With regards to cancer therapy, numerous antitumor agents demonstrate serious side effects (or toxicity), which has led to the unmet need for improving their selectivity and stability. Exosomes, either produced naturally or generated artificially, provide an attractive platform to load many types of molecules such as small molecules, biologics, and other therapeutic agents. Furthermore, the features of exosomes can be designed by selecting their source cells, or they can be engineered to incorporate affinity tags; thus, exosomes show promise as effective delivery vehicles for the complex tumor microenvironment. In this review, we focus on various exosomes produced from different cell types and their potential uses. Moreover, we summarize the current state of artificial exosomes as a drug carrier and provide an overview of the techniques used for their production.

Aberrant Transcript Usage Is Associated with Homologous Recombination Deficiency and Predicts Therapeutic Response.

BRCA1/2 mutations account for only a small fraction of homologous recombination (HR) deficiency (HRD) cases. Recently developed genomic HRD (gHRD) tests suffer confounding factors that cause low precision in predicting samples that will respond to PARP inhibitors and DNA damaging agents. Here we present molecular and clinical evidence of transcriptional HRD (tHRD) that is based on aberrant transcript usage (aTU) of minor isoforms. Specifically, increased TU of nonfunctional isoforms of DNA repair genes was prevalent in breast and ovarian cancer with gHRD. Functional assays validated the association of aTU with impaired HR activity. Machine learning-based tHRD detection by the transcript usage (TU) pattern of key genes was superior to directly screening for gHRD or BRCA1/2 mutations in accurately predicting responses of cell lines and patients with cancer to PARP inhibitors and genotoxic drugs. This approach demonstrated the capability of tHRD status to reflect functional HR status, including in a cohort of olaparib-treated ovarian cancer with acquired platinum resistance. Diagnostic tests based on tHRD are expected to broaden the clinical utility of PARP inhibitors. SIGNIFICANCE: A novel but widespread transcriptional mechanism by which homologous recombination deficiency arises independently of BRCA1/2 mutations can be utilized as a companion diagnostic for PARP inhibitors.

Localized Photothermal Ablation Therapy of Obstructive Rectal Cancer Using a Nanofunctionalized Stent in a Mouse Model.

The self-expanding metal stent (SEMS) is a versatile, palliative treatment method for unresectable, malignant, non-vascular strictures. Colorectal cancer (CRC) is one of the candidates for the application of the SEMS, in combination with the photothermal ablation (PTA) technique that enhances its therapeutic efficacy. The objective of this study was to investigate the efficacy of stent-mediated PTA therapy in an endoscopy-guided, orthotopic rectal cancer model. A total of 30 of 40 mice with the tumor size of grade 4 were included and were divided into three groups of 10 mice each. Group A underwent a gold nanoparticle (AuNP)-coated SEMS but no near-infrared (NIR) irradiation, group B received an uncoated control SEMS with NIR irradiation, and group C received a AuNP-coated SEMS and NIR irradiation together. Colonoscopy and in vivo imaging, immunohistochemical analysis, and quantitative reverse-transcription polymerase chain reaction of major tumor markers were performed. Stent placement and PTA were technically successful using colonoscopy. The tumor grade reduction after PTA is significant in group C, compared with groups A or B (p < 0.001). Molecular analysis validated this observation with a significantly reduced Mapk1 proliferation marker or increased Jnk expression. Histological analysis confirmed the localized PTA therapy using AuNP-coated SEMS profoundly ablated tumor outgrowth through the stent. Our results indicate that this novel strategy of localized PTA therapy could be a promising option for palliative treatment of CRC and to support prolonged stent patency with a decreased tumor volume.

Metagenomic analysis of gut microbiome reveals a dynamic change in Alistipes onderdonkii in the preclinical model of pancreatic cancer, suppressing its proliferation.

Pancreatic cancer is a lethal cancer with aggressive and invasive characteristics. By the time it is diagnosed, patients already have tumors extended to other organs and show extremely low survival rates. The gut microbiome is known to be associated with many diseases and its imbalance affects the pathogenesis of pancreatic cancer. In this study, we established an orthotopic, patient-derived xenograft model to identify how the gut microbiome is linked to pancreatic ductal adenocarcinoma (PDAC). Using the 16S rDNA metagenomic sequencing, we revealed that the levels of Alistipes onderdonkii and Roseburia hominis decreased in the gut microbiome of the PDAC model. To explore the crosstalk between the two bacteria and PDAC cells, we collected the supernatant of the bacteria or cancer cell culture medium and treated it in a cross manner. While the cancer cell medium did not affect bacterial growth, we observed that the A. onderdonkii medium suppressed the growth of the pancreatic primary cancer cells. Using the bromodeoxyuridine/7-amino-actinomycin D (BrdU/7-AAD) staining assay, we confirmed that the A. onderdonkii medium inhibited the proliferation of the pancreatic primary cancer cells. Furthermore, RNA-seq analysis revealed that the A. onderdonkii medium induced unique transcriptomic alterations in the PDAC cells, compared to the normal pancreatic cells. Altogether, our data suggest that the reduction in the A. onderdonkii in the gut microbiome provides a proliferation advantage to the pancreatic cancer cells. KEY POINTS: • Metagenome analysis of pancreatic cancer model reveals A. onderdonkii downregulation. • A. onderdonkii culture supernatant suppressed the proliferation of pancreatic cancer cells. • RNA seq data reveals typical gene expression changes induced by A. onderdonkii.

siRNA Nanoparticle Targeting PD-L1 Activates Tumor Immunity and Abrogates Pancreatic Cancer Growth in Humanized Preclinical Model.

Pancreatic cancer is characterized by late detection, frequent drug resistance, and a highly metastatic nature, leading to poor prognosis. Antibody-based immunotherapy showed limited success for pancreatic cancer, partly owing to the low delivery rate of the drug into the tumor. Herein, we describe a poly(lactic-co-glycolic acid;PLGA)-based siRNA nanoparticle targeting PD-L1 (siPD-L1@PLGA). The siPD-L1@PLGA exhibited efficient knockdown of PD-L1 in cancer cells, without affecting the cell viability up to 6 mg/mL. Further, 99.2% of PDAC cells uptake the nanoparticle and successfully blocked the IFN-gamma-mediated PD-L1 induction. Consistently, the siPD-L1@PLGA sensitized cancer cells to antigen-specific immune cells, as exemplified by Ovalbumin-targeting T cells. To evaluate its efficacy in vivo, we adopted a pancreatic PDX model in humanized mice, generated by grafting CD34+ hematopoeitic stem cells onto NSG mice. The siPD-L1@PLGA significantly suppressed pancreatic tumor growth in this model with upregulated IFN-gamma positive CD8 T cells, leading to more apoptotic tumor cells. Multiplex immunofluorescence analysis exhibited comparable immune cell compositions in control and siPD-L1@PLGA-treated tumors. However, we found higher Granzyme B expression in the siPD-L1@PLGA-treated tumors, suggesting higher activity of NK or cytotoxic T cells. Based on these results, we propose the application of siPD-L1@PLGA as an immunotherapeutic agent for pancreatic cancer.

Identification of a Splice Variant (c.5074+3A>C) of BRCA1 by RNA Sequencing and TOPO Cloning.

Grading the pathogenicity of BRCA1/2 variants has great clinical importance in patient treatment as well as in the prevention and screening of hereditary breast and ovarian cancer (HBOC). For accurate evaluation, confirming the splicing effect of a possible splice site variant is crucial. We report a significant splicing variant (c.5074+3A>C) in BRCA1 in a patient with recurrent ovarian cancer. Next-generation sequencing (NGS) of BRCA1/2 from patient's peripheral blood identified the variant, which was strongly suspected of being a splicing mutation based on in silico predictions. Direct RNA analysis yielded multiple transcripts, and TOPO cloning of the complementary DNA (cDNA) and Sanger sequencing revealed an aberrant transcript with an insertion of the first 153 bp of intron 17, and another transcript with the 153 bp insertion along with an exon 18 deletion. A premature termination codon was presumed to be formed by the 153 bp partial intron retention common to the two transcripts. Therefore, BRCA1 c.5074+3A>C was classified as a likely pathogenic variant. Our findings show that active use of functional studies of variants suspected of altered splicing are of great help in classifying them.

Generation of Colon Cancer Model Based on Colonoscopy Injection.

Colorectal cancer animal model is a very useful tool to explore the tumor initiation and development. In the past year, many methods have been used for building up the mouse model including the subcutaneous injection and cecal wall injection or implantation. But this model cannot reflect the native stromal environment of the colon mucosa. Recently, the in vivo murine endoscopy has been developed allowing high-resolution imaging of the colon. Endoscopy orthotopic injection tumor cell line becomes a low cost, fast tumor growth simple technique. In this chapter, we describe detailed protocols for rapidly and efficiently building up colon cancer tumors model by using the colonoscopy-guided mucosal injection. This model can be used to explore drug testing, gene function assessment, and cancer metastasis.

Targeted eicosanoids profiling reveals a prostaglandin reprogramming in breast Cancer by microRNA-155.

BACKGROUND: Prostaglandin is one of the key metabolites for inflammation-related carcinogenesis. Despite the microRNA-155 is implicated in various types of cancers, it's function in prostaglandin metabolism is largely unknown. METHODS: A targeted profiling of eicosanoids including prostaglandin, leukotriene and thromboxanes was performed in miR-155 deficient breast tumors and cancer cells. The molecular mechanism of miR-155-mediated prostaglandin reprogramming was investigated in primary and cancer cell lines, by analyzing key enzymes responsible for the prostaglandin production. RESULTS: We found miR-155-deficient breast tumors, plasma of tumor-bearing mouse and cancer cells show altered prostaglandin level, especially for the prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2). Subsequent analysis in primary cancer cells, 20 triple-negative breast cancer (TNBC) specimens and breast cancer cell lines with miR-155 knockdown consistently showed a positive correlation between miR-155 level and PGE2/PGD2 ratio. Mechanistically, we reveal the miR-155 reprograms the prostaglandin metabolism by up-regulating PGE2-producing enzymes PTGES/PTGES2 while down-regulating PGD2-producing enzyme PTGDS. Further, we show the up-regulation of PTGES2 is driven by miR-155-cMYC axis, whereas PTGES is transactivated by miR-155-KLF4. Thus, miR-155 hires dual-regulatory mode for the metabolic enzyme expression to reprogram the PGE2/PGD2 balance. Lastly, we show the miR-155-driven cellular proliferation is restored by the siRNA of PTGES1/2, of which expression also significantly correlates with breast cancer patients' survival. CONCLUSIONS: Considering clinical trials targeting PGE2 production largely have focused on the inhibition of Cox1 or Cox2 that showed cardiac toxicity, our data suggest an alternative way for suppressing PGE2 production via the inhibition of miR-155. As the antagomiR of miR-155 (MRG-106) underwent a phase-1 clinical trial, its effect should be considered and analyzed in prostaglandin metabolism in tumor.

The Establishment of a Fast and Safe Orthotopic Colon Cancer Model Using a Tissue Adhesive Technique.

PURPOSE: We aimed to develop a novel method for orthotopic colon cancer model, using tissue adhesive in place of conventional surgical method. MATERIALS AND METHODS: RFP HCT 116 cell line were used to establish the colon cancer model. Fresh tumor tissue harvested from a subcutaneous injection was grafted into twenty nude mice, divided into group A (suture method) and group B (tissue adhesive method). For the group A, we fixed the tissue on the serosa layer of proximal colon by 8-0 surgical suture. For the group B, tissue adhesive (10 µL) was used to fix the tumor. The mortality, tumor implantation success, tumor metastasis, primary tumor size, and operation time were compared between the two groups. Dissected tumor tissue was analyzed for the histology and immunohistochemistry. Also, we performed tumor marker analysis. RESULTS: We observed 30% increase in graft success and 20% decrease in mortality, by using tissue adhesive method, respectively. The median colon tumor size was significantly increased by 4 mm and operation time was shortened by 6.5 minutes. The H&E showed similar tumor structure between the two groups. The immunohistochemistry staining for cancer antigen 19-9, carcinoembryonic antigen, cytokeratin 20, and Ki-67 showed comparable intensities in both groups. Real-time quantitative reverse transcription analysis showed eight out of nine tumor markers are unchanged in the tissue adhesive group. Western blot indicated the tissue adhesive group expressed less p-JNK (apototic marker) and more p-MEK/p-p38 (proliferation marker) levels. CONCLUSION: We concluded the tissue adhesive method is a quick and safe way to generate orthotopic, colon cancer model.

Potential of MALDI-TOF-based serum N-glycan analysis for the diagnosis and surveillance of breast cancer.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based serum N-glycan analysis has gained acknowledgment for the diagnosis of breast cancer in recent years. In this study, the possibilities of expanding its application for breast cancer management and surveillance were discovered and evaluated. First, a novel MALDI-TOF platform, IDsys RT, was confirmed to be effective for breast cancer analysis, showing a maximum area under the curve of 0.91. Multiple N-glycan markers were identified and validated using this process, and they were found to be applicable for differentiating recurring breast cancer samples from healthy control or ordinary breast cancer samples. Recurrence samples were especially distinct from non-recurrence samples when N-glycan signatures were sampled in multiple time points and monitored via MALDI-TOF, throughout the therapy. These results suggested the feasibility of MALDI-TOF-based N-glycan analysis for tracking the molecular signatures of breast cancer and predicting recurrence.