Transcriptome-based deep learning analysis identifies drug candidates targeting protein synthesis and autophagy for the treatment of muscle wasting disorder.

Sarcopenia, the progressive decline in skeletal muscle mass and function, is observed in various conditions, including cancer and aging. The complex molecular biology of sarcopenia has posed challenges for the development of FDA-approved medications, which have mainly focused on dietary supplementation. Targeting a single gene may not be sufficient to address the broad range of processes involved in muscle loss. This study analyzed the gene expression signatures associated with cancer formation and 5-FU chemotherapy-induced muscle wasting. Our findings suggest that dimenhydrinate, a combination of 8-chlorotheophylline and diphenhydramine, is a potential therapeutic for sarcopenia. In vitro experiments demonstrated that dimenhydrinate promotes muscle progenitor cell proliferation through the phosphorylation of Nrf2 by 8-chlorotheophylline and promotes myotube formation through diphenhydramine-induced autophagy. Furthermore, in various in vivo sarcopenia models, dimenhydrinate induced rapid muscle tissue regeneration. It improved muscle regeneration in animals with Duchenne muscular dystrophy (DMD) and facilitated muscle and fat recovery in animals with chemotherapy-induced sarcopenia. As an FDA-approved drug, dimenhydrinate could be applied for sarcopenia treatment after a relatively short development period, providing hope for individuals suffering from this debilitating condition.

Molecular Subtypes Based on Genomic and Transcriptomic Features Correlate with the Responsiveness to Immune Checkpoint Inhibitors in Metastatic Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) has been reported to be highly immune to and infiltrated by T cells and has angiogenesis features, but the effect of given features on clinical outcomes followed by immune checkpoint inhibitors (ICIs) in ccRCC has not been fully characterized. Currently, loss of function mutation in PBRM1, a PBAF-complex gene frequently mutated in ccRCC, is associated with clinical benefit from ICIs, and is considered as a predictive biomarker for response to anti-PD-1 therapy. However, functional mechanisms of PBRM1 mutation regarding immunotherapy responsiveness are still poorly understood. Here, we performed targeted sequencing (n = 60) and whole transcriptomic sequencing (WTS) (n = 61) of patients with metastatic ccRCC treated by ICIs. By integrating WTS data from the CheckMate 025 trial, we obtained WTS data of 177 tumors and finally identified three molecular subtypes that are characterized by distinct molecular phenotypes and frequency of PBRM1 mutations. Patient clustered subtypes 1 and 3 demonstrated worse responses and survival after ICIs treatment, with a low proportion of PBRM1 mutation and angiogenesis-poor, but were immune-rich and cell-cycle enriched. Notably, patients clustered in the subtype 2 showed a better response and survival after ICIs treatment, with enrichment of PBRM1 mutation and metabolic programs and a low exhausted immune phenotype. Further analysis of the subtype 2 population demonstrated that GATM (glycine amidinotransferase), as a novel gene associated with PBRM1 mutation, plays a pivotal role in ccRCC by using a cell culture model, revealing tumor, suppressive-like features in reducing proliferation and migration. In summary, we identified that metastatic ccRCC treated by ICIs have distinct genomic and transcriptomic features that may account for their responsiveness to ICIs. We also revealed that the novel gene GATM can be a potential tumor suppressor and/or can be associated with therapeutic efficacy in metastatic ccRCC treated by ICIs.

Isolation and Genomic Analysis of Single Circulating Tumor Cell Using Human Telomerase Reverse Transcriptase and Desmoglein-2.

As epithelial cells in the circulation are considered to originate from the tumor, the epithelial cell adhesion molecule has been commonly used as a standard marker for circulating tumor cells (CTCs) isolation. However, it seems to disappear after the epithelial-mesenchymal transition that most cancer cells undergo for intravasation. Thus, more advanced techniques for CTC detection are needed to better understand the clinical significance of CTCs. A cancer cell-specifically-infecting or replicating virus that codes a fluorescent monitor gene can be a solution to efficiently detect CTCs. Thus, the authors designed an adenovirus to bind to desmoglein-2, which is highly expressed in most cancer cells. A cancer-specific human telomerase reverse transcriptase promoter is inserted to control a viral E1 region. The adenovirus is utilized to compare the number of CTCs from renal cell carcinoma and prostate cancer patients before and after surgery. The isolated two or three CTCs are eligible for whole genome sequencing. The genomic analysis proves the difference of variants between primary tumors and CTCs. Taken together, it is a fast and exact serial method for CTC isolation and the enriched genome sequencing may be used to determine the prognosis and as a point-of-care system for patients with cancer.

Identification of drug combinations on the basis of machine learning to maximize anti-aging effects.

Aging is a multifactorial process that involves numerous genetic changes, so identifying anti-aging agents is quite challenging. Age-associated genetic factors must be better understood to search appropriately for anti-aging agents. We utilized an aging-related gene expression pattern-trained machine learning system that can implement reversible changes in aging by linking combinatory drugs. In silico gene expression pattern-based drug repositioning strategies, such as connectivity map, have been developed as a method for unique drug discovery. However, these strategies have limitations such as lists that differ for input and drug-inducing genes or constraints to compare experimental cell lines to target diseases. To address this issue and improve the prediction success rate, we modified the original version of expression profiles with a stepwise-filtered method. We utilized a machine learning system called deep-neural network (DNN). Here we report that combinational drug pairs using differential expressed genes (DEG) had a more enhanced anti-aging effect compared with single independent treatments on leukemia cells. This study shows potential drug combinations to retard the effects of aging with higher efficacy using innovative machine learning techniques.

Selective Targeting of Cancer Stem Cells (CSCs) Based on Photodynamic Therapy (PDT) Penetration Depth Inhibits Colon Polyp Formation in Mice.

Targeting cancer stem cells (CSCs) without damaging normal stem cells could contribute to the development of novel radical cancer therapies. Cells expressing leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) constitute a cancer-causing population in the colon; therefore, targeting of Lgr5+ cells is expected to provide an opportunity to mitigate colon cancer. However, the expression of Lgr5 in normal stem cells makes it difficult to prove the efficacy of therapies targeted exclusively at Lgr5+ cancer cells. We used a modified photodynamic therapy technique involving cellular radiative transfer between green fluorescent protein (GFP)-expressing cells and a rose bengal photosensitizer. After treatment, tumors containing GFP-Lgr5+ cells were observed to be significantly suppressed or retarded with little effect on GFP-Lgr5+ stem cells at the crypt bottom. Lgr5+ CSCs were specifically eradicated in situ, when localized based on the depth from the colon lumen, revealing the potential preventive efficacy of Lgr5-targeted therapy on tumor growth. This study supports the idea that Lgr5+ cells localized near the colon luminal surface are central to colorectal cancer. With further development, the targeting of localized Lgr5+ cancer stem cells, which this study demonstrates in concept, may be feasible for prevention of colon cancer in high-risk populations.

Micro-endoscopic In Vivo Monitoring in the Blood and Lymphatic Vessels of the Oral Cavity after Radiation Therapy.

Radiotherapy, although used worldwide for the treatment of head, neck, and oral cancers, causes acute complications, including effects on vasculature and immune response due to cellular stress. Thus, the ability to diagnose side-effects and monitor vascular response in real-time during radiotherapy would be highly beneficial for clinical and research applications. In this study, recently-developed fluorescence micro-endoscopic technology provides non-invasive, high-resolution, real-time imaging at the cellular level. Moreover, with the application of high-resolution imaging technologies and micro-endoscopy, which enable improved monitoring of adverse effects in GFP-expressing mouse models, changes in the oral vasculature and lymphatic vessels are quantified in real time for 10 days following a mild localized single fractionation, 10 Gy radiotherapy treatments. Fluorescence micro-endoscopy enables quantification of the cardiovascular recovery and immune response, which shows short-term reduction in mean blood flow velocity, in lymph flow, and in transient immune infiltration even after this mild radiation dose, in addition to long-term reduction in blood vessel capacity. The data provided may serve as a reference for the expected cellular-level physiological, cardiovascular, and immune changes in animal disease models after radiotherapy.

Correction to: Selective killing of circulating tumor cells prevents metastasis and extends survival.

The original article [1] contains an inadvertent omission in the Funding declaration.

Selective killing of circulating tumor cells prevents metastasis and extends survival.

Distant metastasis is initiated by circulating tumor cells (CTCs), which are considered to be a determining factor for the degree of metastasis and the survival of cancer patients. Although CTC-based diagnostic approaches are being rapidly developed, limited studies have proven the benefits of CTC elimination, with most studies providing only hypothetical inference because of the technical difficulty in examining the effects of CTC elimination in vivo. We modified photodynamic therapy to specifically eliminate green fluorescent protein (GFP)-expressing CTCs and evaluated the therapeutic efficacy of CTC elimination. When circulating blood is illuminated with a blue laser (λ = 473 nm), the combination of GFP and photosensitizers induces a selective elimination of GFP-expressing CTCs, with limited effect on normal cells. In GFP-expressing cancer cell-infused or transplanted mice models, the treatment suppressed distant metastasis and extended the survival of the tumor-bearing mice. Taken together, CTCs are a core seed to be metastasized into secondary organs and elimination of CTCs may improve the survival of cancer patients.

Integrin α6 as an invasiveness marker for hepatitis B viral X-driven hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) accounts for more than 60% of hepatocellular carcinoma (HCC) cases. However, there is limited information about the features of HBV-driven HCC that differentiate it from other types of HCC. OBJECTIVE: The aim of this study is to find a gene specific to HBV-driven HCC and understand its role during tumorigenesis. METHODS: The differences in gene expression patterns were analyzed among patients with hepatitis virus-unrelated liver cirrhosis, and hepatitis C virus- and HBV-driven HCC. Genes expressed only in HBV patients were compared to genes of transgenic mice expressing hepatitis B viral X gene. RESULTS: Integrin α6 was commonly overexpressed in both HBV-driven HCC patients and transgenic mice expressing viral X. This gene's activation induced overexpression of integrin α6, as well as formation of integrins α6ß1 and α6ß4, without changing the expression of non-integrin laminin receptors. Suppression of integrin α6 caused significant inhibition of tumor migration in vitro. CONCLUSIONS: This study found a significant association between HBV and integrin α6, which may be responsible for early migration and invasion of HCC. Thus, integrin α6 is a predictive marker for tumor recurrence and invasiveness of HBV-driven HCC.

Evaluation of pemetrexed and etoposide as therapeutic regimens for human papillomavirus-positive oral and oropharyngeal cancer.

Although human papillomavirus (HPV) positive oral and oropharyngeal cancers have distinct epidemiologic and molecular characteristics compared to HPV-negative cancers, all patients with oral and oropharyngeal cancers received same standard regimen regardless of HPV status. For these reasons, specific regimens for patients with HPV-positive oral and oropharyngeal cancer are needed. Differentially expressed genes (DEG) between HPV-positive and HPV-negative oropharyngeal cancers were re-analyzed and categorized from public database. Then, druggable targets to HPV-positive oral and oropharyngeal cancer were identified and were validated with E6/E7, which is oncogene of HPV, transfected oral and oropharyngeal cancer cell lines and HPV infected cell lines. In DEG analysis, HPV-positive oral and oropharyngeal cancer showed distinct disease entity from HPV-negative cancers. Unlike HPV-negative oral and oropharyngeal cancer, thymidylate synthase (TS) and topoisomerase II (Topo II) were overexpressed in HPV-positive cancers. Transfection of Lenti-virus containing E6/ E7 to HPV-negative oral and oropharyngeal cancer cells induced upregulation of TS and Topo II in those cells. Although cisplatin, which is standard regimen in head and neck cancers, showed more effectiveness in HPV-negative cells, 5-FU and pemetrexed, which are TS inhibitors, or etoposide, which is Topo II inhibitors, worked more effectively in HPV-positive cells. In addition, cisplatin/etoposide and cisplatin/pemetrexed combination regimens showed synergic effects in HPV-positive cells. Pemetrexed or etoposide alone, or in combination with other chemotherapeutic agents such as cisplatin, can be used as novel substitutes in a regimen of concurrent chemoradiotherapy or a palliative regimen for HPV-positive oral and oropharyngeal cancer patients. However, a well-designed clinical trial is needed.

Fluorescent cell-selective ablation using an adaptive photodynamic method.

Intravital ablation of particular cell populations is necessary to decipher their roles under spatiotemporal conditions. Energy transfer-based photodynamic therapy presented a conditional range for specifically inducing the death of GFP expressing cells, with little effect on normal cells. This novel system enables easy access to the functional study of cells.

Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression.

Matrix metalloproteinase (MMP) is a key marker and target molecule for cancer diagnosis, as MMP is able to cleave peptide chains resulting in degradation of extracellular matrix (ECM), a necessary step for cancer development. In particular, MMP2 has recently been recognized as an important biomarker for lung cancer. Despite the important role of detecting MMP molecules in cancer diagnosis, it is a daunting task to quantitatively understand a correlation between the status of cancer development and the secretion level of MMP in a blood droplet. Here, we demonstrate a nanoscale cancer diagnosis by nanomechanical quantitation of MMP2 molecules under cancer progression with using a blood droplet of lung cancer patients. Specifically, we measured the frequency dynamics of nanomechanical biosensor functionalized with peptide chains mimicking ECM in response to MMP2 secreted from tumors in lung with different metastasis level. It is shown that the frequency shift of the biosensor, which exhibits the detection sensitivity below 1 nM, enables the quantitation of the secretion level of MMP2 molecules during the progression of cancer cells or tumor growth. More importantly, using a blood droplet of lung cancer patients, nanomechanical biosensor is shown to be capable of depicting the correlation between the secretion level of MMP2 molecules and the level of cancer metastasis, which highlights the cantilever-based MMP2 detection for diagnosis of lung cancer. Our finding will broaden the understanding of cancer development activated by MMP and allow for a fast and point-of-care cancer diagnostics.

Transcriptome analysis indicates TFEB1 and YEATS4 as regulatory transcription factors for drug resistance of ovarian cancer.

Ovarian cancer is an intractable disease because patients with ovarian cancer frequently develop drug resistance after long-term chemotherapy. Despite the availability of cumulative information on drug-resistant patients, strategies to reverse drug resistance have still not been established. In this study, we analyzed drug resistance-associated transcription factors (TFs) in ovarian cancer. Gene expression profiles of 15 drug-resistant and 11 drug-sensitive patients with ovarian cancer were compared. Our results showed that TFs TFEB1 and YEATS4 regulated the expression of downstream target genes. These 2 TFs have already been implicated in tumorigenesis or metastasis. To our knowledge, this is the first study to evaluate the involvement of these TFs in drug resistance of ovarian cancer. Interestingly, 70% knockdown of each of these TFs with siRNAs resulted in approximately 20%~30% recovery of drug sensitivity. Further, combination treatment of ovarian cancer cells with TFEB1 and YEATS4 siRNAs resulted in 35% reversal of drug resistance. The effect of these TFs on chemoresistance seemed to be associated with intrinsic apoptosis-related pathways, such as p53 activation, and not with the suppression of drug transport. Thus, we suggest a novel approach to reverse chemoresistance of ovarian cancer by suppressing TFEB1 and YEATS4.

Bioluminescence-activated deep-tissue photodynamic therapy of cancer.

Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm(2) for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT.

Gemcitabine plus split-dose cisplatin could be a promising alternative to gemcitabine plus carboplatin for cisplatin-unfit patients with advanced urothelial carcinoma.

PURPOSE: Cisplatin-based chemotherapies are standard treatment regimens of advanced urothelial cell carcinoma. But a significant proportion of patients are unsuitable for cisplatin due to impaired renal function. Carboplatin-based regimens such as gemcitabine and carboplatin regimen (GCb) were applied due to less nephrotoxicity and side effects in these patients. However, it is known that clinical outcome of carboplatin-based regimens was unsatisfactory compared to cisplatin-based regimens. We compared the nephrotoxicity and response to treatment between GCb and gemcitabine plus split-dose cisplatin regimen (GC-S). METHODS: GC-S consists of cisplatin 35 mg/m(2) given on day 1, 2 and gemcitabine 1000 mg/m(2) on day 1, 8 every 3 weeks. GCb consists of carboplatin (AUC 4.5) on day 1 and gemcitabine 1000 mg/m(2) on day 1, 8 every 3 weeks. Patient demographics, serum creatinine and calculated GFR, adverse events, and radiologic response were retrospectively reviewed. RESULTS: Forty-four patients with advanced urothelial carcinoma treated with GCb (n = 22) or GC-S (n = 22) in our institution. There was no difference at deterioration of serum creatinine or GFR between GCb and GC-S (p = 0.442, p = 0.345). For patients who had GFR < 60 mL/min/1.73 m(2) subgroup, similar results were produced (p = 0.292, p = 0.186). In addition, GC-S (68.4 %) showed improved response compared to GCb (31.6 %) (p = 0.023). Both treatments were well tolerated, and there were no unexpected serious adverse events. CONCLUSIONS: Based on preserved renal function, favorable response, and tolerability, GC-S could be a promising alternative to GCb for cisplatin-unfit patients with advanced urothelial carcinoma.

Longitudinal Tracing of Spontaneous Regression and Anti-angiogenic Response of Individual Microadenomas during Colon Tumorigenesis.

Angiogenesis is essential for the progression of cancer, but its involvement in the initial phase of colon tumorigenesis is not well understood. Using intravital endomicroscopy, we visualized the natural history of early pre-tumorous lesions and adenomas in the colon of conditional Apc-knockout and Apc/Kras double mutant mouse models. Early lesions emerged about 4 weeks after the onset of somatic mutations, accompanying vascular dilation when the size of lesions reached about 200 µm, but most lesions regressed spontaneously and cleared within 10 weeks after their emergence. Anti-angiogenic treatments with vascular endothelial growth factor receptor (VEGFR) antagonists reduced the size of the early lesions and the number of polyps. We found surprisingly that anti-angiogenic treatments delayed the natural clearance of transient lesions by up to several weeks in both genetic models. The results represent the previously unexpected role of early angiogenesis on the spontaneous regression of early-stage colon tumors.

In vivo imaging of Lgr5-positive cell populations using confocal laser endomicroscopy during early colon tumorigenesis.

BACKGROUND AND STUDY AIMS: A diagnostic molecular marker for pre-neoplastic lesions, particularly before polyposis, is still lacking. Lgr5 has been broadly accepted as a marker for intestinal cancer stem cells. The aim of this study was to investigate the monitoring of Lgr5( + ) cells as a useful tool for the early diagnosis of premalignant lesions before polyp formation. METHODS: In vivo molecular imaging was performed to examine colon tumorigenesis in Lgr5-EGFP mice treated with azoxymethane and dextran sodium sulfate. eGFP( +) Lgr5( +) regions in the descending colon were longitudinally monitored using side-view confocal endomicroscopy. Based on the eGFP signal intensity on the luminal surface, polyps were classified into two groups - Lgr5-high and Lgr5-low. White light colonoscopy was used to monitor polyp formation. RESULTS: Approximately 75 % of the polyps originated from foci containing Lgr5-eGFP( +) cells, whereas 25 % of the polyps emerged from Lgr5( -) foci. Among eGFP( +) foci, Lgr5-high foci grew faster than Lgr5-low foci. CONCLUSIONS: Polyps developed at Lgr5( +) regions. Luminal Lgr5 expression was correlated with the growth rate of early-stage adenomas. Lgr5 is a promising molecular marker for the early diagnosis of colon tumors.

Bioimaging of hyaluronic acid derivatives using nanosized carbon dots.

Fluorescent nanosized carbon dots (Cdots) are an emerging bioimaging agent with excellent chemical inertness and marginal cytotoxicity in comparison to widely used semiconductor quantum dots. In this work, we report the application of Cdots for real time bioimaging of target specific delivery of hyaluronic acid (HA) derivatives. Polyethylene glycol (PEG) diamine-capped Cdots were synthesized by the pyrolysis of citric acid in a hot solvent. The synthesized Cdots showed strong fluorescence under UV excitation with emission properties dependending on the excitation wavelength. HA-Cdot conjugates were synthesized by amide bond formation between amine groups of Cdot and carboxylic groups of HA. After confirmation of the negligible cytotoxicity of Cdots and HA-Cdot conjugates, in vitro bioimaging was carried out for target specific intracellular delivery of the HA-Cdot conjugates by HA receptor-mediated endocytosis. Furthermore, in vivo real-time bioimaging of Cdots and HA-Cdot conjugates exhibited the target specific delivery of HA-Cdot conjugates to the liver with abundant HA receptors. Taken together, we could confirm the feasibility of HA derivatives as a target-specific drug delivery carrier for the treatment of liver diseases and Cdots as a promising bioimaging agent.

Peroral direct cholangioscopic-guided biopsy and photodynamic therapy using an ultraslim upper endoscope for recurrent hepatocellular carcinoma with intraductal tiny nodular tumor growth.

Bile-duct invasion is rare in patients with hepatocellular carcinoma (HCC). We report a case that received peroral direct cholangioscopy (PDCS)-guided endoscopic biopsy and photodynamic treatment (PDT) for recurrent HCC with intraductal tiny nodular tumor growth. A 64-year-old woman presented with recurrent right upper-quadrant pain. Six months previously she had been diagnosed with HCC with bile-duct invasion in the right anterior segment and had received right anterior segmentectomy. On pathological examination, the margin of resection was clear, but macroscopic bile-duct invasion was noted. On admission, magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography (ERCP) revealed a 0.5-cm-sized polypoid mass at the hilar portion. ERCP-guided biopsy failed, and an ampullary stricture was noted. PDCS-guided endoscopic biopsy was thus performed, and histopathology of the retrieved specimen revealed HCC. The patient submitted to PDT. There was no procedure-related complication. After 1 month of PDT the polypoid lesion and scar change at the hilar lesion had disappeared.