Variations in the cystic duct: frequency and the relationship among insertion sides and heights on the bile duct.

BACKGROUND: Evaluation of the cystic duct anatomy prior to bile duct or gallbladder surgery is important, to decrease the risk of bile duct injury. This study aimed to clarify the frequency of cystic duct variations and the relationship between them. METHODS: Data of 205 patients who underwent cholecystectomy after imaging at Sada Hospital, Japan, were analyzed. The Chi-square test was used to analyze the relationships among variations. RESULTS: The lateral and posterior sides of the bile duct were the two most common insertion points (92 patients, 44.9%), and the middle height was the most common insertion height (135 patients, 65.9%). Clinically important variations (spiral courses, parallel courses, low insertions, and right hepatic duct draining) relating to the risk of bile duct injury were observed in 24 patients (11.7%). Regarding the relationship between the insertion sides and heights, we noticed that the posterior insertion frequently existed in low insertions (75.0%, P < 0.001) and did not exist in high insertions. In contrast, the anterior insertion coexisted with high and never low insertions. Spiral courses have two courses: anterior and posterior, and anterior ones were only found in high insertion cases. CONCLUSIONS: The insertion point of the cystic duct and the spiral courses tended to be anterior or lateral superiorly and posterior inferiorly. Clinically significant variations in cystic duct insertions are common and surgeons should be cautious about these variations to avoid complications.

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.

Tumor- and Nerve-Derived Axon Guidance Molecule Promotes Pancreatic Ductal Adenocarcinoma Progression and Metastasis through Macrophage Reprogramming.

Axon guidance molecules were found to be the gene family most frequently altered in pancreatic ductal adenocarcinoma (PDA) through mutations and copy number changes. However, the exact molecular mechanism regarding PDA development remained unclear. Using genetically engineered mouse models to examine one of the axon guidance molecules, semaphorin 3D (SEMA3D), we found a dual role for tumor-derived SEMA3D in malignant transformation of pancreatic epithelial cells and a role for nerve-derived SEMA3D in PDA development. This was demonstrated by the pancreatic-specific knockout of the SEMA3D gene from the KRAS G12D and TP53 R 172 H mutation knock-in, PDX1-Cre (KPC) mouse model which demonstrated a delayed tumor initiation and growth comparing to the original KPC mouse model. Our results showed that SEMA3D knockout skews the macrophages in the pancreas away from M2 polarization, providing a potential mechanistic role of tumor-derived SEMA3D in PDA development. The KPC mice with the SEMA3D knockout remained metastasis-free, however, died from primary tumor growth. We then tested the hypothesis that a potential compensation mechanism could result from SEMA3D which is naturally expressed by the intratumoral nerves. Our study further revealed that nerve-derived SEMA3D does not reprogram macrophages directly, but reprograms macrophages indirectly through ARF6 signaling and lactate production in PDA tumor cells. SEMA3D increases tumor-secreted lactate which is sensed by GPCR132 on macrophages and subsequently stimulates pro-tumorigenic M2 polarization in vivo. Tumor intrinsic- and extrinsic-SEMA3D induced ARF6 signaling through its receptor Plexin D1 in a mutant KRAS-dependent manner. Consistently, RNA sequencing database analysis revealed an association of higher KRAS MUT expression with an increase in SEMA3D and ARF6 expression in human PDAs. Moreover, multiplex immunohistochemistry analysis showed an increased number of M2-polarized macrophages proximal to nerves in human PDA tissue expressing SEMA3D. Thus, this study suggests altered expression of SEMA3D in tumor cells lead to acquisition of cancer-promoting functions and the axon guidance signaling originating from nerves is "hijacked" by tumor cells to support their growth. Other axon guidance and neuronal development molecules may play a similar dual role which is worth further investigation. One sentence summary: Tumor- and nerve-derived SEMA3D promotes tumor progression and metastasis through macrophage reprogramming in the tumor microenvironment. STATEMENT OF SIGNIFICANCE: This study established the dual role of axon guidance molecule, SEMA3D, in the malignant transformation of pancreatic epithelial cells and of nerve-derived SEMA3D in PDA progression and metastasis. It revealed macrophage reprogramming as the mechanism underlying bothroles. Together, this research elucidated how inflammatory responses promote invasive PDA progression and metastasis through an oncogenic process.

Engineered TCR T-cell therapy targeting mass spectrometry-identified natural epitope in PDAC.

Tumor antigens are crucial targets for T-cell-based therapy to induce tumor-specific rejection. However, identifying pancreatic ductal adenocarcinoma (PDAC)-specific T-cell epitopes has been challenging. Using advanced mass spectrometry (MS) analysis, we previously identified cancer-associated, class I MHC-bound epitopes shared by multiple PDAC patients with different HLA-A types. Here, we investigated one of these epitopes, LAMC2203-211, a naturally occurring nonmutated epitope on the LAMC2 protein. Following stimulation with the LAMC2203-211 peptide, we cloned T-cell receptors (TCRs) and transduced them into the Jurkat human T-cell line using a lentiviral vector. We found that Jurkat cells expressing LAMC2203-211-specific TCRs resulted in potent, LAMC2 specific, in vitro cytotoxic effects on PDAC cells. Furthermore, in mice that harbored either subcutaneously or orthotopically implanted tumors originating from both HLA-A allele-matched and unmatched PDAC patients, tumor growth was suppressed in a LAMC2-dependent manner following the infusion of LAMC2-targeting T cells. We have therefore developed a LAMC2-specific TCR-based T-cell therapy strategy likely suitable for many PDAC patients. This is the first study to adopt MS analysis to identify natural CD8+ T-cell epitopes in PDAC that could potentially serve as targets for PDAC immunotherapy.

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.

CCR2/CCR5 inhibitor permits the radiation-induced effector T cell infiltration in pancreatic adenocarcinoma.

The resistance of pancreatic ductal adenocarcinoma (PDAC) to immune checkpoint inhibitors (ICIs) is attributed to the immune-quiescent and -suppressive tumor microenvironment (TME). We recently found that CCR2 and CCR5 were induced in PDAC following treatment with anti-PD-1 antibody (αPD-1); thus, we examined PDAC vaccine or radiation therapy (RT) as T cell priming mechanisms together with BMS-687681, a dual antagonist of CCR2 and CCR5 (CCR2/5i), in combination with αPD-1 as new treatment strategies. Using PDAC mouse models, we demonstrated that RT followed by αPD-1 and prolonged treatment with CCR2/5i conferred better antitumor efficacy than other combination treatments tested. The combination of RT + αPD-1 + CCR2/5i enhanced intratumoral effector and memory T cell infiltration but suppressed regulatory T cell, M2-like tumor-associated macrophage, and myeloid-derived suppressive cell infiltration. RNA sequencing showed that CCR2/5i partially inhibited RT-induced TLR2/4 and RAGE signaling, leading to decreased expression of immunosuppressive cytokines including CCL2/CCL5, but increased expression of effector T cell chemokines such as CCL17/CCL22. This study thus supports the clinical development of CCR2/5i in combination with RT and ICIs for PDAC treatment.

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.

Cancer-associated fibroblast heterogeneity is associated with organ-specific metastasis in pancreatic ductal adenocarcinoma.

BACKGROUND: Metastasis occurs in the majority of pancreatic ductal adenocarcinoma (PDAC) patients at diagnosis or following resection. Patients with liver metastasis and those with lung metastasis have significantly different prognosis. Here, we sought to understand how cancer-associated fibroblasts (CAFs) play roles in the development of organ-specific metastasis. METHODS: PDAC tumor cell lines established from the primary tumors with liver and lung metastasis potentials, respectively, in Kras/p53 mutation conditional knock-in (KPC) mice were co-cultured with matched CAFs or mouse mesenchymal stem cells. CAFs were isolated from metastases and subjected to DNA methylation and whole transcriptomic RNA sequencing analysis. RESULTS: The ability of mouse PDAC tumor cell lines in developing liver or lung-specific metastases was demonstrated in orthotopic models. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induced the methylation of metabolism genes including NQO1 and ALDH1a3 and subsequent downregulated mRNA expression of a broader group of metabolism genes in CAFs. DNA methylation and downregulation of metabolism genes in CAFs in liver metastasis, but not those in lung metastasis, appeared to be regulated by DNA methyltransferase. Tumor cells associated with liver metastasis potential, but not those associated with lung metastasis potential, induce inflammatory CAF (iCAF) signatures. CAFs from liver metastasis demonstrated a more homogenous iCAF phenotype, whereas CAFs from lung metastasis maintained the heterogeneity. CONCLUSIONS: PDAC with organ-specific metastatic potentials has different capacities in inducing methylation of metabolism genes in CAFs, modulating CAF phenotypes, and resulting in different levels of heterogeneity of CAFs in different metastatic niches.

Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism.

How tumor-associated macrophages transit from a predominant antitumor M1-like phenotype to a protumoral M2-like phenotype during the development of pancreatic ductal adenocarcinoma (PDA) remains to be elucidated. We thus conducted a study by employing a PDA-macrophage co-culture system, an "orthotopic" PDA syngeneic mouse model, and human PDA specimens, together with macrophages derived from GARP knockout mice and multiple analytic tools including whole-genome RNA sequencing, DNA methylation arrays, multiplex immunohistochemistry, metabolism measurement, and invasion/metastasis assessment. Our study showed that PDA tumor cells, through direct cell-cell contact, induce DNA methylation and downregulation of a panel of glucose metabolism and OXPHOS genes selectively in M1-like macrophages, leading to a suppressed glucose metabolic status in M1-like but not in M2-like macrophages. Following the interaction with PDA tumor cells, M1-like macrophages are reprogrammed phenotypically to M2-like macrophages. The interaction between M1-like macrophages and PDA cells is mediated by GARP and integrin αV/ß8, respectively. Blocking either GARP or integrin would suppress tumor-induced DNA methylation in Nqo-1 gene and the reprogramming of M1-like macrophages. Glucose-response genes such as Il-10 are subsequently activated in tumor-educated M1-like macrophages. Partly through Il-10 and its receptor Il-10R on tumor cells, M1-like macrophages functionally acquire a pro-cancerous capability. Both exogenous M1-like and M2-like macrophages promote metastasis in a mouse model of PDA while such a role of M1-like macrophages is dependent on DNA methylation. Our results suggest that PDA cells are able to reprogram M1-like macrophages metabolically and functionally through a GARP-dependent and DNA methylation-mediated mechanism to adopt a pro-cancerous fate.

Usefulness of several factors and clinical scoring models in preoperative diagnosis of complicated appendicitis.

BACKGROUND: The preoperative distinction between uncomplicated and complicated appendicitis is important to determine the appropriate treatments, such as antibiotics, surgery, or interval appendectomy. Computed tomography (CT) plays an important role; however, combining clinical and imaging factors may make preoperative evaluation more reliable. This study evaluated and analyzed cases and the usefulness of several preoperative factors and clinical scoring models to detect complicated appendicitis. METHODS: A total of 203 patients preoperatively diagnosed with acute appendicitis at our facility were included. Complicated appendicitis was defined as appendicitis with gangrene, perforated appendix, and/or abscess formation. Preoperative factors were collected from published clinical scoring models; patient information, symptoms, signs, results of laboratory tests, and findings of CT. Factors were analyzed using a chi-squared test and the Mann-Whitney U test. RESULTS: The preoperative factors were compared between 151 uncomplicated and 52 complicated appendicitis patients. The significant factors were age ≥40, duration of symptoms >24 hours, body temperature ≥37.3°C, high levels of CRP, findings in CT scan (appendix diameter ≥10 mm, stranding of the adjacent fat, presence of fluid collection, and suspicion of abscess or perforation). We also evaluated the usefulness of clinical scoring models for the detection of complicated appendicitis and found the Appendicitis Inflammatory Response score and two prediction models (Atema score and Imaoka score) showed significance (p < 0.05). High serum CRP level was significantly associated with complicated appendicitis (p < 0.001), and the predicted existence rates of complicated appendicitis were 52.7% for serum CRP level ≥50mg/L, 74.4% for ≥100mg/L, and 82.6% for ≥150mg/L. CONCLUSION: The results demonstrated several preoperative factors and clinical scoring models to increase suspicion of complicated appendicitis. Specifically, high serum levels of CRP may be a useful factor in predicting complicated appendicitis prior to surgery when supported by clinical findings and imaging; however, further research is needed.

Effect of gallbladder polyp size on the prediction and detection of gallbladder cancer.

BACKGROUND: Gallbladder polyps are relatively common. Although most gallbladder polyps are benign, some are malignant. Current guidelines state that malignancy should be suspected for polyps ≥ 10 mm in diameter. We clarified the cancer detection rates in accordance with the size distribution of gallbladder polyps, and evaluated the effectiveness of the reported risk factors in predicting malignancy. METHODS: In this retrospective case-control study, our institutional database was searched to identify patients who underwent laparoscopic cholecystectomy for benign or malignant gallbladder polyps at Sada Hospital, Japan. The chi-squared test was used to analyze the risk factors for malignancy. RESULTS: There were 227 protruding gallbladder lesions. The 206 benign polyps had a diameter of 2-21 mm, while the 21 malignant polyps were 7-60 mm. The cancer detection rates were 16.4% for lesions ≥ 10 mm, 55.9% for lesions ≥ 15 mm, and 94.1% for lesions ≥ 20 mm. Of the benign lesions, cholesterol polyps were the most frequent (50-100%) in all size ranges, even in large lesions (≥ 15 mm). The sessile lesion morphology was significantly more frequent in malignant (60%) than benign lesions (3.4%, p < 0.00001). Multiple polyps were frequently diagnosed not only as cholesterol polyps (81.1%), but also as adenomas (60%); adenomas were found as a single adenoma within other types of polyps. There were two cases of malignant small gallbladder polyps (< 10 mm); these lesions met the surgical indications of a size increase during observation or a sessile morphology. CONCLUSIONS: The cancer detection rate increased significantly with an increase in the lesion size. Risk factors such as a sessile polyp morphology or an increase in lesion size were effective in predicting malignancy for small gallbladder polyps. It might be difficult to accurately predict the pathologic diagnoses of gallbladder polyps preoperatively, as cholesterol polyps were most frequent, even in the large size range.

Preoperative diagnoses and identification rates of unexpected gallbladder cancer.

BACKGROUND: Unexpected gallbladder cancer (UGBC) is sometimes found in the resected gallbladder of patients during or after surgery. Some reports have indicated UGBC identification rates for all gallbladder surgeries, but scarce data are available for the UGBC identification rates for specific gallbladder diseases. The present study was performed to clarify UGBC rates and the factors suspicious for UGBC categorized according to preoperative diagnoses, in patients undergoing laparoscopic cholecystectomy (LSC). METHODS: We recorded data for all LSC surgeries performed in the Department of Surgery, Sada Hospital, Japan since 1991, and analyzed the 28-year data. We used the chi-square test and Kaplan-Meier analysis for this retrospective case-control study. RESULTS: The UGBC identification rate was 0.69% (63/9186 patients). The UGBC identification rates categorized according to the preoperative diagnoses were 1.3% (13/969) for acute cholecystitis, 2.4% (16/655) for benign tumor, 2.0% (28/1383) for chronic cholecystitis or cholecystitis, and 0.054% (3/5585) for cholecystolithiasis. The percentage of older patients (≥ 60 years) was significantly higher in UGBCs compared with cases finally diagnosed as benign in each group categorized according to the preoperative diagnoses (p≤0.0014), except for cholecystolithiasis. In cases pre-diagnosed as benign tumor, UGBCs were associated with higher rates of thickened gallbladder wall compared with benign tumor (69.2% vs. 27.9%, respectively; p = 0.0011). UGBCs pre-diagnosed as acute cholecystitis had higher T2-T4 rates (100% vs. 64.3%, respectively; p<0.05) and lower survival rates (p = 0.0149) than UGBCs pre-diagnosed with chronic cholecystitis. CONCLUSIONS: UGBC identification rates depend on the preoperative diagnosis and range from 0.054% to 2.4%. Older age (≥ 60 years) could be related to UGBC, and a pre-diagnosis of acute cholecystitis might indicate more advanced cancer compared with a pre-diagnosis of chronic cholecystitis.

Interrogating the immune-modulating roles of radiation therapy for a rational combination with immune-checkpoint inhibitors in treating pancreatic cancer.

BACKGROUND: Radiation therapy (RT) has the potential to enhance the efficacy of immunotherapy, such as checkpoint inhibitors, which has dramatically altered the landscape of treatments for many cancers, but not yet for pancreatic ductal adenocarcinoma (PDAC). Our prior studies demonstrated that PD ligand-1 and indoleamine 2,3-dioxygenase 1 (IDO1) were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT, suggesting RT may prime PDAC for PD-1 blockade antibody (αPD-1) or IDO1 inhibitor (IDO1i) treatments. In this study, we investigated the antitumor efficacy of the combination therapies with radiation and PD-1 blockade or IDO1 inhibition or both. METHODS: We developed and used a mouse syngeneic orthotopic model of PDAC suitable for hypofractionated RT experiments. RESULTS: The combination therapy of αPD-1 and RT improved survival. The dual combination of RT/IDO1i and triple combination of RT/αPD-1/IDO1i did not improve survival compared with RT/αPD-1, although all of these combinations offer similar local tumor control. RT/αPD-1 appeared to result in the best systemic interferon-γ response compared with other treatment groups and the highest local expression of immune-activation genes, including Cd28 and Icos. CONCLUSION: Our RT model allows examining the immune-modulatory effects of RT alone and in combination with immune-checkpoint inhibitors in the pancreas/local microenvironment. This study highlights the importance of choosing the appropriate immune-modulatory agents to be combined with RT to tip the balance toward antitumor adaptive immune responses.

Axon Guidance Molecules Promote Perineural Invasion and Metastasis of Orthotopic Pancreatic Tumors in Mice.

BACKGROUND & AIMS: Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice. METHODS: We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves. RESULTS: DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. CONCLUSIONS: DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.

Extradomain-B Fibronectin-Targeted Dextran-Based Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Probe for Detecting Pancreatic Cancer.

A dextran-peptide conjugate was developed for magnetic resonance (MR) molecular imaging of pancreatic ductal adenocarcinoma (PDAC) through its overexpressed microenvironment biomarker, extradomain-B fibronectin (EDB-FN). This new agent consists of diamagnetic and biocompatible dextran and a targeting peptide. Dextrans can be directly detected by chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) without the need for radionuclide or metallic labeling. In addition, large molecular weight dextran, dextran 10 (MW ∼ 10 kDa), provides an approximately 50 times higher sensitivity per molecule than a single glucose unit. The potential of this highly biocompatible diamagnetic probe is demonstrated in a murine syngeneic allograft PDAC tumor model. The biocompatibility and sensitivity of this new agent clearly show potential for a path to clinical translation.

DNA methylation in the tumor microenvironment.

The tumor microenvironment (TME) plays an important role in supporting cancer progression. The TME is composed of tumor cells, the surrounding tumor-associated stromal cells, and the extracellular matrix (ECM). Crosstalk between the TME components contributes to tumorigenesis. Recently, one of our studies showed that pancreatic ductal adenocarcinoma (PDAC) cells can induce DNA methylation in cancer-associated fibroblasts (CAFs), thereby modifying tumor-stromal interactions in the TME, and subsequently creating a TME that supports tumor growth. Here we summarize recent studies about how DNA methylation affects tumorigenesis through regulating tumor-associated stromal components including fibroblasts and immune cells. We also discuss the potential for targeting DNA methylation for the treatment of cancers.

Cancer-Associated Fibroblasts in Pancreatic Cancer Are Reprogrammed by Tumor-Induced Alterations in Genomic DNA Methylation.

Stromal fibrosis is a prominent histologic characteristic of pancreatic ductal adenocarcinoma (PDAC), but how stromal fibroblasts are regulated in the tumor microenvironment (TME) to support tumor growth is largely unknown. Here we show that PDAC cells can induce DNA methylation in cancer-associated fibroblasts (CAF). Upon direct contact with PDAC cells, DNA methylation of SOCS1 and other genes is induced in mesenchymal stem cells or in CAF that lack SOCS1 methylation at baseline. Silencing or decitabine treatment to block the DNA methylation enzyme DNMT1 inhibited methylation of SOCS1. In contrast, SOCS1 gene methylation and downregulation in CAF activated STAT3 and induced insulin-like growth factor-1 expression to support PDAC cell growth. Moreover, CAF facilitated methylation-dependent growth of PDAC tumor xenografts in mice. The ability of patient-derived CAF with SOCS1 methylation to promote PDAC growth was more robust than CAF without SOCS1 methylation. Overall, our results reveal how PDAC cells can reprogram CAF to modify tumor-stromal interactions in the TME, which promote malignant growth and progression. Cancer Res; 76(18); 5395-404. ©2016 AACR.

Maintenance of algal endosymbionts in Paramecium bursaria: a simple model based on population dynamics.

Algal endosymbiosis is widely distributed in eukaryotes including many protists and metazoans, and plays important roles in aquatic ecosystems, combining phagotrophy and phototrophy. To maintain a stable symbiotic relationship, endosymbiont population size in the host must be properly regulated and maintained at a constant level; however, the mechanisms underlying the maintenance of algal endosymbionts are still largely unknown. Here we investigate the population dynamics of the unicellular ciliate Paramecium bursaria and its Chlorella-like algal endosymbiont under various experimental conditions in a simple culture system. Our results suggest that endosymbiont population size in P. bursaria was not regulated by active processes such as cell division coupling between the two organisms, or partitioning of the endosymbionts at host cell division. Regardless, endosymbiont population size was eventually adjusted to a nearly constant level once cells were grown with light and nutrients. To explain this apparent regulation of population size, we propose a simple mechanism based on the different growth properties (specifically the nutrient requirements) of the two organisms, and based from this develop a mathematical model to describe the population dynamics of host and endosymbiont. The proposed mechanism and model may provide a basis for understanding the maintenance of algal endosymbionts.

Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification.

Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.

TM4SF1 as a prognostic marker of pancreatic ductal adenocarcinoma is involved in migration and invasion of cancer cells.

The cell surface protein Transmembrane 4 L6 family member 1 (TM4SF1) has been detected in various tumors, and its expression on tumor cells is implicated in cancer cell metastasis and patient prognosis. The role of TM4SF1 in malignant tumors remains poorly understood, particularly in pancreatic cancer. We performed immunohistochemical staining to analyze the expression of TM4SF1 in resected pancreatic tissues and investigated the correlation between TM4SF1 expression and prognosis. The function of TM4SF1 in the invasion and migration of pancreatic cancer cells was analyzed in vitro using an RNA interference technique. In pancreatic cancer tissues, TM4SF1 expression was detected in cancer cells, and patients with high tumor levels of TM4SF1 showed longer survival times than those with low TM4SF1 levels (P=0.0332). In vitro, reduced TM4SF1 expression enhanced the migration (P<0.05) and invasion (P<0.05) of pancreatic cancer cells partially via decreased E-cadherin expression. TM4SF1 protein levels were also reduced after TGF-ß1-induced epithelial-mesenchymal transition (EMT).TM4SF1 expression is associated with better prognosis in pancreatic cancer. Loss of TM4SF1 contributes to the invasion and migration of pancreatic cancer cells.