Unenriched xylem water contribution during cellulose synthesis influenced by atmospheric demand governs the intra-annual tree-ring δ18 O signature.

The oxygen isotope composition (δ18 O) of tree-ring cellulose is used to evaluate tree physiological responses to climate, but their interpretation is still limited due to the complexity of the isotope fractionation pathways. We assessed the relative contribution of seasonal needle and xylem water δ18 O variations to the intra-annual tree-ring cellulose δ18 O signature of larch trees at two sites with contrasting soil water availability in the Swiss Alps. We combined biweekly δ18 O measurements of soil water, needle water, and twig xylem water with intra-annual δ18 O measurements of tree-ring cellulose, xylogenesis analysis, and mechanistic and structural equation modeling. Intra-annual cellulose δ18 O values resembled source water δ18 O mean levels better than needle water δ18 O. Large parts of the rings were formed under high proportional exchange with unenriched xylem water (pex ). Maximum pex values were achieved in August and imprinted on sections at 50-75% of the ring. High pex values were associated with periods of high atmospheric evaporative demand (VPD). While VPD governed needle water δ18 O variability, we estimated a limited Péclet effect at both sites. Due to a variable pex , source water has a strong influence over large parts of the intra-annual tree-ring cellulose δ18 O variations, potentially masking signals coming from needle-level processes.

Role of drug catabolism, modulation of oncogenic signaling and tumor microenvironment in microbe-mediated pancreatic cancer chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) has one of the highest incidence/death ratios among all neoplasms due to its late diagnosis and dominant chemoresistance. Most PDAC patients present with an advanced disease characterized by a multifactorial, inherent and acquired resistance to current anticancer treatments. This remarkable chemoresistance has been ascribed to several PDAC features including the genetic landscape, metabolic alterations, and a heterogeneous tumor microenvironment that is characterized by dense fibrosis, and a cellular contexture including functionally distinct subclasses of cancer-associated fibroblasts, immune suppressive cells, but also a number of bacteria, shaping a specific tumor microbiome microenvironment. Thus, recent studies prompted the emergence of a new research avenue, by describing the role of the microbiome in gemcitabine resistance, while next-generation-sequencing analyses identified a specific microbiome in different tumors, including PDAC. Functionally, the contribution of these microbes to PDAC chemoresistance is only beginning to be explored. Here we provide an overview of the studies demonstrating that bacteria have the capacity to metabolically transform and hence inactivate anticancer drugs, as exemplified by the inhibition of the efficacy of 10 out of 30 chemotherapeutics by Escherichia coli. Moreover, a number of bacteria modulate specific oncogenic pathways, such as Fusobacterium nucleatum, affecting autophagy and apoptosis induction by 5-fluorouracil and oxaliplatin. We hypothesize that improved understanding of how chemoresistance is driven by bacteria could enhance the efficacy of current treatments, and discuss the potential of microbiome modulation and targeted therapeutic approaches as well as the need for more reliable models and biomarkers to translate the findings of preclinical/translational research to the clinical setting, and ultimately overcome PDAC chemoresistance, hence improving clinical outcome.

Differential Sensitivity to Ionizing Radiation in Gemcitabine-Resistant and Paclitaxel-Resistant Pancreatic Cancer Cells.

PURPOSE: Chemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). Although chemoradiation has proven effective in other tumor types, such as head and neck squamous cell carcinoma, its role in PDAC and effect on acquired chemoresistance have yet to be fully explored. In this study, we investigated the sensitivity of gemcitabine-resistant (GR) and paclitaxel-resistant (PR) PDAC cells to ionizing radiation (IR) and their underlying mechanisms. METHODS AND MATERIALS: GR and PR clones were generated from PANC-1, PATU-T, and SUIT2-007 pancreatic cancer cell lines. Cell survival after radiation was assessed using clonogenic assay, sulforhodamine B assay, apoptosis, and spheroid growth by bioluminescence. Radiation-induced DNA damage was assessed using Western blot, extra-long polymerase chain reaction, reactive oxygen species production, and immunofluorescence. Autophagy and modulation of the Hippo signaling pathway were investigated using proteomics, Western blot, immunofluorescence, and reverse-transcription quantitative polymerase chain reaction. RESULTS: In both 2- and 3-dimensional settings, PR cells were more sensitive to IR and showed decreased ß-globin amplification, indicating more DNA damage accumulation compared with GR or wild-type cells after 24 hours. Proteomic analysis of PR PATU-T cells revealed that the protein MST4, a kinase involved in autophagy and the Hippo signaling pathway, was highly downregulated. A differential association was found between autophagy and radiation treatment depending on the cell model. Interestingly, increased yes-associated protein nuclear localization and downstream Hippo signaling pathway target gene expression were observed in response to IR. CONCLUSIONS: This was the first study investigating the potential of IR in targeting PDAC cells with acquired chemoresistance. Our results demonstrate that PR cells exhibit enhanced sensitivity to IR due to greater accumulation of DNA damage. Additionally, depending on the specific cellular context, radiation-induced modulation of autophagy and the Hippo signaling pathway emerged as potential underlying mechanisms, findings with potential to inform personalized treatment strategies for patients with acquired chemoresistance.

ABCB1 overexpression through locus amplification represents an actionable target to combat paclitaxel resistance in pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it. METHODS: Three paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance. RESULTS: Upregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel. CONCLUSION: Upregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC.

Prognostic Significance of Integrin Subunit Alpha 2 (ITGA2) and Role of Mechanical Cues in Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma (PDAC).

INTRODUCTION: PDAC is an extremely aggressive tumor with a poor prognosis and remarkable therapeutic resistance. The dense extracellular matrix (ECM) which characterizes PDAC progression is considered a fundamental determinant of chemoresistance, with major contributions from mechanical factors. This study combined biomechanical and pharmacological approaches to evaluate the role of the cell-adhesion molecule ITGA2, a key regulator of ECM, in PDAC resistance to gemcitabine. METHODS: The prognostic value of ITGA2 was analysed in publicly available databases and tissue-microarrays of two cohorts of radically resected and metastatic patients treated with gemcitabine. PANC-1 and its gemcitabine-resistant clone (PANC-1R) were analysed by RNA-sequencing and label-free proteomics. The role of ITGA2 in migration, proliferation, and apoptosis was investigated using hydrogel-coated wells, siRNA-mediated knockdown and overexpression, while collagen-embedded spheroids assessed invasion and ECM remodeling. RESULTS: High ITGA2 expression correlated with shorter progression-free and overall survival, supporting its impact on prognosis and the lack of efficacy of gemcitabine treatment. These findings were corroborated by transcriptomic and proteomic analyses showing that ITGA2 was upregulated in the PANC-1R clone. The aggressive behavior of these cells was significantly reduced by ITGA2 silencing both in vitro and in vivo, while PANC-1 cells growing under conditions resembling PDAC stiffness acquired resistance to gemcitabine, associated to increased ITGA2 expression. Collagen-embedded spheroids of PANC-1R showed a significant matrix remodeling and spreading potential via increased expression of CXCR4 and MMP2. Additionally, overexpression of ITGA2 in MiaPaCa-2 cells triggered gemcitabine resistance and increased proliferation, both in vitro and in vivo, associated to upregulation of phospho-AKT. CONCLUSIONS: ITGA2 emerged as a new prognostic factor, highlighting the relevance of stroma mechanical properties as potential therapeutic targets to counteract gemcitabine resistance in PDAC.

Biodegradable Ultrasmall-in-Nano Architectures Loaded with Cisplatin Prodrug in Combination with Ionizing Radiation Induces DNA Damage and Apoptosis in Pancreatic Ductal Adenocarcinoma.

Considering the dismal survival rate, novel therapeutic strategies are warranted to improve the outcome of pancreatic ductal adenocarcinoma (PDAC). Combining nanotechnology for delivery of chemotherapeutics-preferably radiosensitizing agents-is a promising approach to enhance the therapeutic efficacy of chemoradiation. We assessed the effect of biodegradable ultrasmall-in-nano architectures (NAs) containing gold ultra-small nanoparticles (USNPs) enclosed in silica shells loaded with cisplatin prodrug (NAs-cisPt) combined with ionizing radiation (IR). The cytotoxic effects and DNA damage induction were evaluated in PDAC cell lines (MIA PaCa2, SUIT2-028) and primary culture (PDAC3) in vitro and in the chorioallantoic membrane (CAM) in ovo model. Unlike NAs, NAs-cisPt affected the cell viability in MIA PaCa2 and SUIT2-028 cells. Furthermore, NAs-cisPt showed increased γH2AX expression up to 24 h post-IR and reduced ß-globin amplifications resulting in apoptosis induction at DNA and protein levels. Similarly, combined treatment of NAs-cisPt + IR in PDAC3 and SUIT2-028 CAM models showed enhanced DNA damage and apoptosis leading to tumor growth delay. Our results demonstrate an increased cytotoxic effect of NAs-cisPt, particularly through its release of the cisplatin prodrug. As cisplatin is a well-known radiosensitizer, administration of cisplatin prodrug in a controlled fashion through encapsulation is a promising new treatment approach which merits further investigation in combination with other radiosensitizing agents.

Dynamic Evaluation of Circulating miRNA Profile in EGFR-Mutated NSCLC Patients Treated with EGFR-TKIs.

BACKGROUND: Resistance to EGFR-TKIs constitutes a major challenge for the management of EGFR-mutated NSCLC, and recent evidence suggests that deregulation of specific microRNAs (miRNAs) may influence resistance to targeted agents. In this retrospective study, we explored the role of specific plasmatic miRNAs (miR-21, miR-27a and miR-181a) as a surrogate for predicting EGFR-TKI performance in EGFR-mutated NSCLC patients. METHODS: Plasma samples of 39 advanced EGFR-mutated NSCLC patients treated with EGFR-TKIs were collected at different points in time and miRNA levels were assessed by RT-PCR. RESULTS: Higher basal values of miR-21 were reported in patients who achieved a partial/complete response (PR/CR) compared to those with stability/progression of disease (SD/PD) (p = 0.011). Along the same line, patients who experienced a clinical benefit lasting at least six months displayed higher basal levels of circulating miR-21 (p = 0.039). However, dynamic evaluation of miRNA values after two months from the start of EGFR-TKI treatment showed that patients who experienced SD had an increase in miR-21 levels (Fold Change [FC] = 2.6) compared to patients achieving PR/CR (p = 0.029). The same tendency was observed for miR-27a (FC = 3.1) and miR-181a (FC = 2.0), although without reaching statistical significance. Remarkably, preclinical studies showed an increase in miR-21 levels in NSCLC cells that became resistant after exposure to EGFR-TKIs. CONCLUSIONS: Our study provides interesting insights on the role of circulating miRNAs, in particular miR-21, and their dynamic change over time in predicting EGFR-TKI response in EGFR-mutated NSCLC.

Pancreatic cancer resistance conferred by stellate cells: looking for new preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor response to chemo- and (modest-dose conventionally fractionated) radio-therapy. Emerging evidence suggests that pancreatic stellate cells (PSCs) secrete deoxycytidine, which confers resistance to gemcitabine. In particular, deoxycytidine was detected by analysis of metabolites in fractionated media from different mouse PSCs, showing that it caused PDAC cells chemoresistance by reducing the capacity of deoxycytidine kinase (dCK) for gemcitabine phosphorylation. However, data on human models are missing and dCK expression was not associated with clinical efficacy of gemcitabine. We recently established co-culture models of hetero-spheroids including primary human PSCs and PDAC cells showing their importance as a platform to test the effects of cancer- and stroma-targeted drugs. Here, we discuss the limitations of previous studies and the potential use of above-mentioned models to study molecular mechanisms underlying chemo- and radio-resistance.

The Role of the Microbiome in Cancer and Therapy Efficacy: Focus on Lung Cancer.

The microbiome is extremely important for human health; more recently its role in the context of cancer became clear. Microbial effects range from enhancing cancer immunity and cancer therapy efficacy, to promoting cancer progression and inhibiting treatment efficacy. These broad implications led researchers to investigate these specific interactions, as well as how modification of the microbiome can improve cancer survival and treatment efficacy. While these interactions are better established for cancers such as gastric cancer, they are far less understood in others. As non-small cell lung cancer (NSCLC) makes up the majority of lung cancer cases, and is among the top causes of cancer deaths worldwide, understanding the mechanisms by which the microbiome may impact progression and treatment is crucial to improve patient survival and treatment response. A literature review was conducted to reveal the crosslink between human microbiome and lung cancer. This includes immune priming, induction of pro- or anti-tumor response, and the local effects of intra-tumoral microbiota. Overall, this is a complex multifactorial relationship, and there are broad implications as to how this knowledge can improve cancer treatment. Solutions include manipulation of the microbiome using probiotics, bacterial vaccines and antibiotics. Bacteria biomarkers may also be used as a diagnostic tool.

"Open Sesame?": Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analogue, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal analysis of hENT-1 status evaluated its predictive role by both immunohistochemistry (with five different antibodies), and quantitative-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels observed with this antibody were associated with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this analysis and lists molecular factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.

Platelet nitric oxide production and IR: relation with obesity and hypertriglyceridemia.

BACKGROUND AND AIM: Three NOS isoforms are responsible for nitric oxide production in various tissues. Endothelial constitutive NOS is expressed in vascular endothelium and in platelets, contributing to vascular tone regulation and platelet aggregation. The aim of the present work was to examine eNOS polymorphism, to find a correlation with platelet NO production and degree of insulin resistance (IR) in non-diabetic subjects and in patients affected by type 2 diabetes. METHODS AND RESULTS: Seventy-one non-diabetic subjects and 37 patients affected by Type 2 diabetes were recruited. The subjects were subdivided into 3 groups as cut-off for the definition of an insulin resistant state: IR non-diabetic subjects, insulin sensitive subjects, and insulin-resistant patients affected by Type 2 diabetes. Plasma glyco-metabolic parameters, platelet nitric oxide production, endothelial nitric oxide synthase (eNOS) gene polymorphism were measured in all subjects enrolled. Significant differences between groups were found in BMI, fasting glycaemia, fructosamine and HbA(1c), triglycerides and HDL cholesterol levels. Evaluating all the subjects, platelet NO production was significantly related with BMI, waist circumference, and triglycerides concentrations, thus suggesting an association between increased platelet NO production, obesity and hypertriglyceridemia, independent of the degree of insulin-resistance. CONCLUSION: The modified platelet NO synthesis does not seem to be due to eNOS Glu298Asp polymorphism, while it can be hypothesized that it is caused by an iNOS induction, present in obesity, hypertriglyceridemia and in type 2 diabetes.