Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nitric oxide-targeted therapy inhibits stemness and increases the efficacy of tamoxifen in estrogen receptor-positive breast cancer cells.

Cancer stem cells (CSCs) are involved in the resistance of estrogen (ER)-positive breast tumors against endocrine therapy. On the other hand, nitric oxide (NO) plays a relevant role in CSC biology, although there are no studies addressing how this important signaling molecule may contribute to resistance to antihormonal therapy in ER+ breast cancer. Therefore, we explored whether targeting NO in ER+ breast cancer cells impacts CSC subpopulation and sensitivity to hormonal therapy with tamoxifen. NO was targeted in ER+ breast cancer cells by specific NO depletion and NOS2 silencing and mammosphere formation capacity, stem cell markers and tamoxifen sensitivity were analyzed. An orthotopic breast tumor model in mice was also performed to analyze the efficacy of NO-targeted therapy plus tamoxifen. Kaplan-Meier curves were made to analyze the association of NOS2 gene expression with survival of ER+ breast cancer patients treated with tamoxifen. Our results show that targeting NO inhibited mamosphere formation, CSC markers expression and increased the antitumoral efficacy of tamoxifen in ER+ breast cancer cells, whereas tamoxifen-resistant cells displayed higher expression levels of NOS2 and Notch-1 compared with parental cells. Notably, NO-targeted therapy plus tamoxifen was more effective than either treatment alone in an orthotopic breast tumor model in immunodeficient mice. Furthermore, low NOS2 expression was significantly associated with a higher metastasis-free survival in ER+ breast cancer patients treated with tamoxifen. In conclusion, our data support that NO-targeted therapy in ER+ breast cancer may contribute to increase the efficacy of antihormonal therapy avoiding the development of resistance to these treatments.

Humanized Mice for the Study of Immuno-Oncology.

Immunotherapy is revolutionizing cancer treatment; however, complete responses are achieved in only a small fraction of patients and tumor types. Thus, there is an urgent need for predictive preclinical models to drive rational immunotherapeutic drug development, treatment combinations, and to minimize failures in clinical trials. Humanized mouse models (HIS) have been developed to study and modulate the interactions between immune components and tumors of human origin. In this review, we discuss recent advances in the 'humanization' of mouse models to improve the quality of human immune cell reconstitution. We also highlight new insights into the basic mechanisms, and provide a preclinical evaluation of onco-immunotherapies, as well as the limitations thereof, which constitute drivers for the improvement of the models to increase their translational power.

SWATH-based proteomics reveals processes associated with immune evasion and metastasis in poor prognosis colorectal tumours.

Newly emerged proteomic methodologies, particularly data-independent acquisition (DIA) analysis-related approaches, would improve current gene expression-based classifications of colorectal cancer (CRC). Therefore, this study was aimed to identify protein expression signatures using SWATH-MS DIA and targeted data extraction, to aid in the classification of molecular subtypes of CRC and advance in the diagnosis and development of new drugs. For this purpose, 40 human CRC samples and 7 samples of healthy tissue were subjected to proteomic and bioinformatic analysis. The proteomic analysis identified three different molecular CRC subtypes: P1, P2 and P3. Significantly, P3 subtype showed high agreement with the mesenchymal/stem-like subtype defined by gene expression signatures and characterized by poor prognosis and survival. The P3 subtype was characterized by decreased expression of ribosomal proteins, the spliceosome, and histone deacetylase 2, as well as increased expression of osteopontin, SERPINA 1 and SERPINA 3, and proteins involved in wound healing, acute inflammation and complement pathway. This was also confirmed by immunodetection and gene expression analyses. Our results show that these tumours are characterized by altered expression of proteins involved in biological processes associated with immune evasion and metastasis, suggesting new therapeutic options in the treatment of this aggressive type of CRC.

Expression of the Short Form of RON/STK in Feline Mammary Carcinoma.

RON is a tyrosine kinase receptor activated by the macrophage-stimulating protein (MSP) ligand that is overexpressed in human breast cancer. In humans, RON protein can be present in different isoforms, and the most studied isoform is represented by the short form of RON ( sf-RON), which is generated by an alternative promoter located in intron 10 of the RON complementary DNA (cDNA). It plays an important role in breast cancer progression. Considering the many similarities between feline mammary carcinoma (FMC) and human breast cancer, the aim of this study was to investigate the expression of both RON and MSP in FMCs and to identify the presence of the sf-RON transcript. Tissue samples of spontaneous mammary tumors were collected from 60 queens (10 benign lesions, 50 carcinomas). All of the samples were tested for RON and MSP expression by immunohistochemistry; moreover, RNA was extracted from paraffin-embedded tissue samples, and the cDNA was tested by reverse transcription-polymerase chain reaction (RT-PCR) to identify the presence of sf-RON. Immunohistochemistry detected the expression of RON and MSP in 34 of 50 (68%) and 29 of 50 (58%) FMCs, respectively. RT-PCR revealed the presence of the short-form in 18 of 47 (38%) FMCs. This form originates, as in humans, from an alternative promoter (P2), and it codes for the proper feline short form ( sf-RON). sf-RON expression was associated with poorly differentiated tumors and with a shorter disease-free ( P < .05; hazard ratio [HR], 2.2) period and a shorter survival ( P < .05; HR, 2.2). These results support FMC as a suitable model in comparative oncology and identify sf-RON expression as potential predictor of outcomes for this disease.

A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer.

BACKGROUND: Nitric oxide (NO) has been highlighted as an important agent in cancer-related events. Although the inducible nitric oxide synthase (iNOS) isoform has received most attention, recent studies in the literature indicate that the endothelial isoenzyme (eNOS) can also modulate different tumor processes including resistance, angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology and mesenchymal tumors is unknown. RESULTS: Here, we show that eNOS was significantly upregulated in VilCre ERT2 Apc fl/+ and VilCre ERT2 Apc fl/fl mouse intestinal tissue, with intense immunostaining in hyperproliferative crypts. Similarly, the more invasive VilCre ERT2 Apc fl/+ Pten fl/+ mouse model showed an overexpression of eNOS in intestinal tumors whereas this isoform was not expressed in normal tissue. However, none of the three models showed iNOS expression. Notably, when 40 human colorectal tumors were classified into different clinically relevant molecular subtypes, high eNOS expression was found in the poor relapse-free and overall survival mesenchymal subtype, whereas iNOS was absent. Furthermore, Apc fl/fl organoids overexpressed eNOS compared with wild-type organoids and NO depletion with the scavenger carboxy-PTIO (c-PTIO) decreased the proliferation and the expression of stem-cell markers, such as Lgr5, Troy, Vav3, and Slc14a1, in these intestinal organoids. Moreover, specific NO depletion also decreased the expression of CSC-related proteins in human colorectal cancer cells such as ß-catenin and Bmi1, impairing the CSC phenotype. To rule out the contribution of iNOS in this effect, we established an iNOS-knockdown colorectal cancer cell line. NO-depleted cells showed a decreased capacity to form tumors and c-PTIO treatment in vivo showed an antitumoral effect in a xenograft mouse model. CONCLUSION: Our data support that eNOS upregulation occurs after Apc loss, emerging as an unexpected potential new target in poor-prognosis mesenchymal colorectal tumors, where NO scavenging could represent an interesting therapeutic alternative to targeting the CSC subpopulation.

Demethylating and anti-hepatocarcinogenic potential of hesperidin, a natural polyphenol of Citrus juices.

Hepatocellular carcinoma (HCC) is a neoplasia representing the fifth most common malignancy worldwide and the third cause of death from cancer. Diets with high content in fruits and vegetables are widely recommended for their health-promoting properties, among them, the protection against diabetes, cancer, and cardiovascular diseases. Hesperidin is the most important phenol in the orange fruit with well-known health benefits. Diet components have been used as possible modulator agents of DNA methylation in cancer cells and epigenetic therapy against their harmful effects could be a potential tool in chemotherapy. The purpose of the present study was to evaluate the methylation patterns induced by hesperidin in HL60 cell line as an in vitro model in order to analyze its chemopreventive effects in epigenetic cancer therapies. A parallel in vivo pilot experience using a rat diethyl nitrosamine hepatocarcinogenesis-induced model was carried out to validate the therapeutic efficacy of this orange flavonol. Results showed that: (i) Hesperidin is cytotoxic in a dose-dependent manner and the IC50 was 12.5 mM; (ii) Hesperidin exerts a significant hypomethylating effect on the LINE-1 sequence (up to 47% hypomethylation at 12.5 mM) and on the ALU-M2 repetitive sequences (up to 32% at 6 mM) in HL60 tumor cells. (iii) Hesperidin does not affect the rat body and liver weight and it is able to reduce the diethyl nitrosamine-induced nodules at 1,000, 500, and 250 ppm. In conclusion, hesperidin could be proposed as a candidate molecule in chemoprevention in epigenetic therapy purposes.

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma.

BACKGROUND: Progesterone receptors play a key role in the development of canine mammary tumours, and recent research has focussed on their possible value as therapeutic targets using antiprogestins. Cloning and sequencing of the progesterone receptor gene has shown that the receptor has two isoforms, A and B, transcribed from a single gene. Experimental studies in human breast cancer suggest that the differential expression of progesterone receptor isoforms has implications for hormone therapy responsiveness. This study examined the effects of the antiprogestin aglepristone on cell proliferation and mRNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n = 22) or vehicle (n = 5) twice before surgery. RESULTS: Formalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor and both isoforms by RT-qPCR and Ki67 antigen labelling. Both total progesterone receptor and isoform A mRNA expression levels decreased after treatment with aglepristone. Furthermore, a significant decrease in the proliferation index (percentage of Ki67-labelled cells) was observed in progesterone-receptor positive and isoform-A positive tumours in aglepristone-treated dogs. CONCLUSIONS: These findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A.

Primary central nervous system T-cell lymphoma mimicking meningoencephalomyelitis in a cat.

A cat was presented with right head tilt and circling. The lack of expression of virus antigens did not support the postmortem diagnosis of encephalomyelitis pointing to a diffuse primary central nervous system T-cell lymphoma on the basis of CD3 and CD45R co-expression with absence of CD79α staining.

Lymphome primaire de système nerveux central type T imitant méningo-encéphalomyélite chez un chat. Un chat est venu avec inclinaison de la tête à droite et circling. L'absence d'expression des antigènes du virus ne prend pas en charge le diagnositic post mortem e d'une encéphalomyélite pointant vers un lymphome primaire du système nerveux central type T diffus sur la base de CD3 et CD45R coexpression avec absence CD79α expression.(Traduit par les auteurs).

S-Nitrosylation at the intersection of metabolism and autophagy: Implications for cancer.

Metabolic plasticity, which determines tumour growth and metastasis, is now understood to be a flexible and context-specific process in cancer metabolism. One of the major pathways contributing to metabolic adaptations in eucaryotic cells is autophagy, a cellular degradation and recycling process that is activated during periods of starvation or stress to maintain metabolite and biosynthetic intermediate levels. Consequently, there is a close association between the metabolic adaptive capacity of tumour cells and autophagy-related pathways in cancer. Additionally, nitric oxide regulates protein function and signalling through S-nitrosylation, a post-translational modification that can also impact metabolism and autophagy. The primary objective of this review is to provide an up-to-date overview of the role of S-nitrosylation at the intersection of metabolism and autophagy in cancer. First, we will outline the involvement of S-nitrosylation in the metabolic adaptations that occur in tumours. Then, we will discuss the multifaceted role of autophagy in cancer, the interplay between metabolism and autophagy during tumour progression, and the contribution of S-nitrosylation to autophagic dysregulation in cancer. Finally, we will present insights into relevant therapeutic aspects and discuss prospects for the future.

Analysis of cell-free DNA concentration, fragmentation patterns and TP53 gene expression in mammary tumor-bearing dogs: A pilot study.

Introduction: Liquid biopsy based on the analysis of circulating cell-free DNA (cfDNA), as well as on detection of point mutations by digital droplet PCR (ddPCR), has revolutionized the research in oncology. In recent years, this technique has been pioneering in veterinary medicine since it is a minimally invasive approach with very promising results for characterization of tumors. Methods: The aim of this study was, firstly, to analyze the concentration and the fragmentation pattern of cfDNA of dogs with mammary tumors (n = 36) and healthy dogs (n = 5) and its correlation with clinicopathological data. Secondly, analysis of TP53 gene expression and the point mutation in the codon 245 were performed in cfDNA and in tumor tissues to assess their potential as plasma biomarkers. Results and discussion: Our results highlighted that those dogs with worse clinicopathological characteristics (simple or undifferentiated carcinomas, higher histological grade and presence of peritumoral inflammation) shown higher cfDNA concentration and higher concentrations of short-fragments (<190 bp) than healthy dogs. In addition, although no detection of the point mutation in codon 245 of TP53 gene could be detected neither in plasma nor tumor tissue, an increased TP53 expression was detected in animals with tumors bearing malignant characteristics. Finally, a high concordance with TP53 gene expression in plasma and tumor tissue and cfDNA concentration was also found. The results derived from this work confirm the valuable potential of cfDNA and its fragments, as well as the analysis of TP53 expression in plasma as useful liquid biomarkers for clinical application in veterinary oncology.

Circulating NPTX2 methylation as a non-invasive biomarker for prognosis and monitoring of metastatic pancreatic cancer.

BACKGROUND: Pancreatic cancer is the most lethal cancer with a dismal prognosis mainly due to diagnosis at advanced stage and ineffective treatments. CA19-9 levels and computed tomography (CT) imaging are the main standard criteria for evaluating disease progression and treatment response. In this study we explored liquid biopsy-based epigenetic biomarkers for prognosis and monitoring disease in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). METHODS: Plasma samples were collected from 44 mPDAC patients at the time of diagnosis, and in 15 of them, additional samples were obtained during follow-up of the disease. After cell-free DNA (cfDNA), isolation circulating levels of methylated NPTX2, SPARC, BMP3, SFRP1 and TFPI2 genes were measured using digital droplet PCR (ddPCR). BEAMing technique was performed for quantitation of RAS mutations in cfDNA, and CA19-9 was measured using standard techniques. RESULTS: NPTX2 was the most highly and frequently methylated gene in cfDNA samples from mPDAC patients. Higher circulating NPTX2 methylation levels at diagnosis were associated with poor prognosis and efficiently stratified patients for prediction of overall survival (6.06% cut-off, p = 0.0067). Dynamics of circulating NPTX2 methylation levels correlated with disease progression and response to therapy and predicted better than CA19-9 the evolution of disease in mPDAC patients. Remarkably, in many cases the disease progression detected by CT scan was anticipated by an increase in circulating NPTX2 methylation levels. CONCLUSIONS: Our study supports circulating NPTX2 methylation levels as a promising liquid biopsy-based clinical tool for non-invasive prognosis, monitoring disease evolution and response to treatment in mPDAC patients.

Clinical significance of glycogen synthase kinase 3 (GSK-3) expression and tumor budding grade in colorectal cancer: Implications for targeted therapy.

INTRODUCTION: Glycogen synthase kinase 3 (GSK-3) has been proposed as a novel cancer target due to its regulating role in both tumor and immune cells. However, the connection between GSK-3 and immunoevasive contexture, including tumor budding (TB) has not been previously examined. METHODS: we investigated the expression levels of total GSK-3 as well as its isoforms (GSK-3ß and GSK-3α) and examined their potential correlation with TB grade and the programmed cell death-ligand 1 (PD-L1) in colorectal cancer (CRC) tumor samples. Additionally, we compared the efficacy of GSK-3-inhibition with PD-1/PD-L1 blockade in humanized patient-derived (PDXs) xenografts models of high-grade TB CRC. RESULTS: we show that high-grade (BD3) TB CRC is associated with elevated expression levels of total GSK-3, specifically the GSK-3ß isoform, along with increased expression of PD-L1 in tumor cells. Moreover, we define an improved risk stratification of CRC patients based on the presence of GSK-3+/PD-L1+/BD3 tumors, which are associated with a worse prognosis. Significantly, in contrast to the PD-L1/PD-1 blockade approach, the inhibition GSK-3 demonstrated a remarkable enhancement in the antitumor response. This was achieved through the reduction of tumor buds via necrosis and apoptosis pathways, along with a notable increase of activated tumor-infiltrating CD8+ T cells, NK cells, and CD4- CD8- T cells. CONCLUSIONS: our study provides compelling evidence for the clinical significance of GSK-3 expression and TB grade in risk stratification of CRC patients. Moreover, our findings strongly support GSK-3 inhibition as an effective therapy specifically targeting high-grade TB in CRC.

The Combination of Neutrophil-Lymphocyte Ratio and Platelet-Lymphocyte Ratio with Liquid Biopsy Biomarkers Improves Prognosis Prediction in Metastatic Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a highly inflammatory microenvironment and liquid biopsy has emerged as a promising tool for the noninvasive analysis of this tumor. In this study, plasma was obtained from 58 metastatic PDAC patients, and neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), circulating cell-free DNA (cfDNA) concentration, and circulating RAS mutation were determined. We found that NLR was significantly associated with both overall survival (OS) and progression-free survival. Remarkably, NLR was an independent risk factor for poor OS. Moreover, NLR and PLR positively correlated, and combination of both inflammatory markers significantly improved the prognostic stratification of metastatic PDAC patients. NLR also showed a positive correlation with cfDNA levels and RAS mutant allelic fraction (MAF). Besides, we found that neutrophil activation contributed to cfDNA content in the plasma of metastatic PDAC patients. Finally, a multi-parameter prognosis model was designed by combining NLR, PLR, cfDNA levels, RAS mutation, RAS MAF, and CA19-9, which performs as a promising tool to predict the prognosis of metastatic PDAC patients. In conclusion, our study supports the idea that the use of systemic inflammatory markers along with circulating tumor-specific markers may constitute a valuable tool for the clinical management of metastatic PDAC patients.

Up-Regulation of Immune Checkpoints in the Thymus of PRRSV-1-Infected Piglets in a Virulence-Dependent Fashion.

Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.

Immunoneutralization of inhibin in cycling rats increases follicle-stimulating hormone secretion, stimulates the ovary and attenuates progesterone receptor-dependent preovulatory luteinizing hormone secretion.

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Circulating Cell-Free DNA-Based Liquid Biopsy Markers for the Non-Invasive Prognosis and Monitoring of Metastatic Pancreatic Cancer.

Liquid biopsy may assist in the management of cancer patients, which can be particularly applicable in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the utility of circulating cell-free DNA (cfDNA)-based markers as prognostic tools in metastatic PDAC. Plasma was obtained from 61 metastatic PDAC patients, and cfDNA levels and fragmentation were determined. BEAMing technique was used for quantitative determination of RAS mutation allele fraction (MAF) in cfDNA. We found that the prognosis was more accurately predicted by RAS mutation detection in plasma than in tissue. RAS mutation status in plasma was a strong independent prognostic factor for both overall survival (OS) and progression-free survival (PFS). Moreover, RAS MAF in cfDNA was also an independent risk factor for poor OS, and was strongly associated with primary tumours in the body/tail of the pancreas and liver metastases. Higher cfDNA levels and fragmentation were also associated with poorer OS and shorter PFS, body/tail tumors, and hepatic metastases, whereas cfDNA fragmentation positively correlated with RAS MAF. Remarkably, the combination of CA19-9 with MAF, cfDNA levels and fragmentation improved the prognostic stratification of patients. Furthermore, dynamics of RAS MAF better correlated with patients' outcome than standard CA19-9 marker. In conclusion, our study supports the use of cfDNA-based liquid biopsy markers as clinical tools for the non-invasive prognosis and monitoring of metastatic PDAC patients.

Association of Tumor Budding With Immune Evasion Pathways in Primary Colorectal Cancer and Patient-Derived Xenografts.

Tumor budding has been found to be of prognostic significance for several cancers, including colorectal cancer (CRC). Additionally, the molecular classification of CRC has led to the identification of different immune microenvironments linked to distinct prognosis and therapeutic response. However, the association between tumor budding and the different molecular subtypes of CRC and distinct immune profiles have not been fully elucidated. This study focused, firstly, on the validation of derived xenograft models (PDXs) for the evaluation of tumor budding and their human counterparts and, secondly, on the association between tumor budding and the immune tumor microenvironment by the analysis of gene expression signatures of immune checkpoints, Toll-like receptors (TLRs), and chemokine families. Clinical CRC samples with different grades of tumor budding and their corresponding PDXs were included in this study. Tumor budding grade was reliably reproduced in early passages of PDXs, and high-grade tumor budding was intimately related with a poor-prognosis CMS4 mesenchymal subtype. In addition, an upregulation of negative regulatory immune checkpoints (PDL1, TIM-3, NOX2, and IDO1), TLRs (TLR1, TLR3, TLR4, and TLR6), and chemokine receptors and ligands (CXCR2, CXCR4, CXCL1, CXCL2, CXCL6, and CXCL9) was detected in high-grade tumor budding in both human samples and their corresponding xenografts. Our data support a close link between high-grade tumor budding in CRC and a distinctive immune-suppressive microenvironment promoting tumor invasion, which may have a determinant role in the poor prognosis of the CMS4 mesenchymal subtype. In addition, our study demonstrates that PDX models may constitute a robust preclinical platform for the development of novel therapies directed against tumor budding in CRC.

Activation of estrogen receptor-alpha induces gonadotroph progesterone receptor expression and action differently in young and middle-aged ovariectomized rats.

BACKGROUND: We attempted to define the effect of estrogen receptor (ER)alpha activation on gonadotroph progesterone receptor (PR) expression (mRNA and protein) and action (GnRH-stimulated and GnRH self-priming) in short- and long-term ovariectomized (OVX) rats. METHODS: Two weeks or 1 year after OVX, rats were injected over 3 days with 125 microg/kg of estradiol benzoate (EB), 7.5 mg/kg of the selective ERalpha agonist propylpyrazole triol (PPT), or 15 mg/kg of the selective ER modulator tamoxifen (TX). Controls were given 0.2 ml oil. The last day of ER analog treatment, half of the rats in each group received 25 mg/kg of progesterone (P). The next day, anterior pituitaries were removed and analyzed for PR-AB mRNA and protein. Gonadotrophin secretion in incubated pituitaries was also measured. RESULTS: (i) PR mRNA expression was higher in young than in middle-aged OVX rats although PR protein was absent in pituitaries from both groups of OVX rats; (ii) activation of ERalpha reduced gonadotroph hypertrophy and increased PR mRNA and protein expression (EB > PPT > TX) more efficiently in young than in middle-aged rats, (iii) ER agonists elicited GnRH-stimulated LH and FSH secretion in young but only FSH secretion in middle-aged OVX rats, (iv) evaluated by peak LH concentrations, GnRH self-priming was observed in both groups of OVX rats and (v) P down-regulated PR protein expression in young, and to a lesser extent, in middle-aged OVX rats, in close association with PR-dependent GnRH self-priming. CONCLUSIONS: Middle-aged OVX rats exhibited clear-cut LH, but not FSH, secretory defects in pituitary sensitivity to estrogen and P.

Ovarian stimulation with FSH reduces phosphorylation of gonadotrope progesterone receptor and LH secretion in the rat.

Administration of human FSH (hFSH) to cyclic rats during the dioestrous phase attenuates progesterone receptor (PR)-dependent events of the preovulatory LH surge in pro-oestrus. The increased bioactivity of the putative ovarian gonadotropin surge inhibiting/attenuating factor induced by hFSH treatment is not associated with a decrease in PR protein expression, and the possibility of its association at a PR posttranslational effect has been raised. The present experiments aimed to analyse PR phosphorylation status in the gonadotrope of rats with impaired LH secretion induced by in vivo hFSH injection. Two experimental approaches were used. First, incubated pro-oestrous pituitaries from hFSH-injected cycling and oestrogen-treated ovariectomized (OVX) rats were used to analyze the effect of calyculin, an inhibitor of intracellular phosphatases, on PR-dependent LH release, which was measured in the incubation medium by RIA. Second, pituitaries taken from hFSH-injected intact cycling and OVX rats and later incubated with P or GNRH1 were used to assess the phosphorylation rate of gonadotrope. The latter was analysed in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry using a MAB that recognizes the phosphorylated (p) form of PR at Ser294. Calyculin reduced the ovary-mediated inhibition of hFSH in GNRH1-stimulated LH secretion. In addition, the immunohistochemical expression of pSer294 PR was significantly reduced after ovarian stimulation with hFSH in pituitaries from pro-oestrous rats incubated with P or GNRH1. Altogether, these results suggested that the ovarian-dependent inhibitory effect of FSH injection on the preovulatory LH secretion in the rat may involve an increase in dephosphorylation of PR.