Mature tertiary lymphoid structures are key niches of tumour-specific immune responses in pancreatic ductal adenocarcinomas.

OBJECTIVE: To better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity. DESIGN: We characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial. RESULTS: We found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-ß may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expanded CXCL13 + tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens. CONCLUSION: We provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

MEL-Index: Estimation of Tissue Melatonin Levels Using Gene Expression Data.

Melatonin synthesis by extrapineal sources adjusts physiological and pathophysiological processes in several types of cells and tissues. As measuring locally produced melatonin in fresh tissues might be a challenge due to limited material availability, we created a simple predictive model, the MEL-Index, which infers the content of tissue melatonin using gene expression data. The MEL-Index can be a powerful tool to study the role of melatonin in different contexts. Applying the MEL-Index method to RNA-seq datasets, we have shed light into the clinical relevance of melatonin as a modulator tumor progression and lung infection due to COVID-19. The MEL-Index combines the z-normalized expressions of ASMT (Acetylserotonin O-Methyltransferase), last enzyme of the biosynthetic pathway, and CYP1B1 (cytochrome P450 family enzyme), which encodes the enzyme that metabolizes melatonin in extrahepatic tissues. In this chapter, we describe the steps for calculating the MEL-Index.

Melanopsin (Opn4) is an oncogene in cutaneous melanoma.

The search for new therapeutical targets for cutaneous melanoma and other cancers is an ongoing task. We expanded this knowledge by evaluating whether opsins, light- and thermo-sensing proteins, could display tumor-modulatory effects on melanoma cancer. Using different experimental approaches, we show that melanoma cell proliferation is slower in the absence of Opn4, compared to Opn4WT due to an impaired cell cycle progression and reduced melanocyte inducing transcription factor (Mitf) expression. In vivo tumor progression of Opn4KO cells is remarkably reduced due to slower proliferation, and higher immune system response in Opn4KO tumors. Using pharmacological assays, we demonstrate that guanylyl cyclase activity is impaired in Opn4KO cells. Evaluation of Tumor Cancer Genome Atlas (TCGA) database confirms our experimental data as reduced MITF and OPN4 expression in human melanoma correlates with slower cell cycle progression and presence of immune cells in the tumor microenvironment (TME). Proteomic analyses of tumor bulk show that the reduced growth of Opn4KO tumors is associated with reduced Mitf signaling, higher translation of G2/M proteins, and impaired guanylyl cyclase activity. Conversely, in Opn4WT tumors increased small GTPase and an immune-suppressive TME are found. Such evidence points to OPN4 as an oncogene in melanoma, which could be pharmacologically targeted.

B Cell Orchestration of Anti-tumor Immune Responses: A Matter of Cell Localization and Communication.

The immune system plays a crucial role in cancer development either by fostering tumor growth or destroying tumor cells, which has open new avenues for cancer immunotherapy. It was only over the last decade that the role of B cells in controlling anti-tumor immune responses in the tumor milieu has begun to be appreciated. B and plasma cells can exert anti-tumor effects through antibody-dependent cell cytotoxicity (ADCC) and activation of the complement cascade, even though their effector functions extend beyond the classical humoral immunity. In tumor tissues, B cells can be found in lymphoid aggregates, known as tertiary lymphoid structures (TLSs), well-organized non-encapsulated structures composed of immune and stromal cells. These structures reflect a process of lymphoid neogenesis occurring in peripheral tissues upon long-lasting exposure to inflammatory signals. The TLS provides an area of intense B cell antigen presentation that can lead to optimal T cell activation and effector functions, as well as the generation of effector B cells, which can be further differentiated in either antibody-secreting plasma cells or memory B cells. Of clinical interest, the crosstalk between B cells and antigen-experienced and exhausted CD8+ T cells within mature TLS was recently associated with improved response to immune checkpoint blockade (ICB) in melanoma, sarcoma and lung cancer. Otherwise, B cells sparsely distributed in the tumor microenvironment or organized in immature TLSs were found to exert immune-regulatory functions, inhibiting anti-tumor immunity through the secretion of anti-inflammatory cytokines. Such phenotype might arise when B cells interact with malignant cells rather than T and dendritic cells. Differences in the spatial distribution likely underlie discrepancies between the role of B cells inferred from human samples or mouse models. Many fast-growing orthotopic tumors develop a malignant cell-rich bulk with reduced stroma and are devoid of TLSs, which highlights the importance of carefully selecting pre-clinical models. In summary, strategies that promote TLS formation in close proximity to tumor cells are likely to favor immunotherapy responses. Here, the cellular and molecular programs coordinating B cell development, activation and organization within TLSs will be reviewed, focusing on their translational relevance to cancer immunotherapy.

An Immunometabolic Shift Modulates Cytotoxic Lymphocyte Activation During Melanoma Progression in TRPA1 Channel Null Mice.

Melanoma skin cancer is extremely aggressive with increasing incidence and mortality. Among the emerging therapeutic targets in the treatment of cancer, the family of transient receptor potential channels (TRPs) has been reported as a possible pharmacological target. Specifically, the ankyrin subfamily, representing TRPA1 channels, can act as a pro-inflammatory hub. These channels have already been implicated in the control of intracellular metabolism in several cell models, but little is known about their role in immune cells, and how it could affect tumor progression in a process known as immune surveillance. Here, we investigated the participation of the TRPA1 channel in the immune response against melanoma tumor progression in a mouse model. Using Trpa1 +/+ and Trpa1 -/- animals, we evaluated tumor progression using murine B16-F10 cells and assessed isolated CD8+ T cells for respiratory and cytotoxic functions. Tumor growth was significantly reduced in Trpa1 -/- animals. We observed an increase in the frequency of circulating lymphocytes. Using a dataset of CD8+ T cells isolated from metastatic melanoma patients, we found that TRPA1 reduction correlates with several immunological pathways. Naïve CD8+ T cells from Trpa1 +/+ and Trpa1 -/- animals showed different mitochondrial respiration and glycolysis profiles. However, under CD3/CD28 costimulatory conditions, the absence of TRPA1 led to an even more extensive metabolic shift, probably linked to a greater in vitro killling ability of Trpa1 -/- CD8+ T cells. Therefore, these data demonstrate an unprecedented role of TRPA1 channel in the metabolism control of the immune system cells during carcinogenesis.

Melanopsin mediates UVA-dependent modulation of proliferation, pigmentation, apoptosis, and molecular clock in normal and malignant melanocytes.

Cutaneous melanocytes and melanoma cells express several opsins, of which melanopsin (OPN4) detects temperature and UVA radiation. To evaluate the interaction between OPN4 and UVA radiation, normal and malignant Opn4WT and Opn4KO melanocytes were exposed to three daily low doses (total 13.2 kJ/m2) of UVA radiation. UVA radiation led to a reduction of proliferation in both Opn4WT cell lines; however, only in melanoma cells this effect was associated with increased cell death by apoptosis. Daily UVA stimuli induced persistent pigment darkening (PPD) in both Opn4WT cell lines. Upon Opn4 knockout, all UVA-induced effects were lost in three independent clones of Opn4KO melanocytes and melanoma cells. Per1 bioluminescence was reduced after 1st and 2nd UVA radiations in Opn4WT cells. In Opn4KO melanocytes and melanoma cells, an acute increase of Per1 expression was seen immediately after each stimulus. We also found that OPN4 expression is downregulated in human melanoma compared to normal skin, and it decreases with disease progression. Interestingly, metastatic melanomas with low expression of OPN4 present increased expression of BMAL1 and longer overall survival. Collectively, our findings reinforce the functionality of the photosensitive system of melanocytes that may subsidize advancements in the understanding of skin related diseases, including cancer.

The balance between NRF2/GSH antioxidant mediated pathway and DNA repair modulates cisplatin resistance in lung cancer cells.

Lung cancer patients face a dismal prognosis mainly due to the low efficacy of current available treatments. Cisplatin is the first-line chemotherapy treatment for those patients, however, resistance to this drug is a common and yet not fully understood phenomenon. Aiming to shed new light into this puzzle, we used established normal and malignant lung cell lines displaying different sensitivity towards cisplatin treatment. We observed a negative correlation between cell viability and DNA damage induction upon cisplatin treatment. Interestingly, drug sensitivity in those cell lines was not due to either difference on DNA repair capacity, or in the amount of membrane ion channel commonly used for cisplatin uptake. Also, we noted that glutathione intracellular levels, and expression and activity of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) were determinant for cisplatin cytotoxicity. Remarkably, analysis of gene expression in non-small cell lung cancer patients of the TCGA data bank revealed that there is a significant lower overall survival rate in the subset of patients bearing tumors with unbalanced levels of NRF2/KEAP1 and, as consequence, increased expression of NRF2 target genes. Thus, the results indicate that NRF2 and glutathione levels figure as important cisplatin resistance biomarkers in lung cancer.

ATR mediates cisplatin resistance in 3D-cultured breast cancer cells via translesion DNA synthesis modulation.

Tissue architecture and cell-extracellular matrix (cell-ECM) interaction determine the organ specificity; however, the influences of these factors on anticancer drugs preclinical studies are highly neglected. For considering such aspects, three-dimensional (3D) cell culture models are relevant tools for accurate analysis of cellular responses to chemotherapy. Here we compared the MCF-7 breast cancer cells responses to cisplatin in traditional two-dimensional (2D) and in 3D-reconstituted basement membrane (3D-rBM) cell culture models. The results showed a substantial increase of cisplatin resistance mediated by 3D microenvironment. This phenotype was independent of p53 status and autophagy activity and was also observed for other cellular models, including lung cancer cells. Such strong decrease on cellular sensitivity was not due to differences on drug-induced DNA damage, since similar levels of γ-H2AX and cisplatin-DNA adducts were detected under both conditions. However, the processing of these cisplatin-induced DNA lesions was very different in 2D and 3D cultures. Unlike cells in monolayer, cisplatin-induced DNA damage is persistent in 3D-cultured cells, which, consequently, led to high senescence induction. Moreover, only 3D-cultured cells were able to progress through S cell cycle phase, with unaffected replication fork progression, due to the upregulation of translesion (TLS) DNA polymerase expression and activation of the ATR-Chk1 pathway. Co-treatment with VE-821, a pharmacological inhibitor of ATR, blocked the 3D-mediated changes on cisplatin response, including low sensitivity and high TLS capacity. In addition, ATR inhibition also reverted induction of REV3L by cisplatin treatment. By using REV3L-deficient cells, we showed that this TLS DNA polymerase is essential for the cisplatin sensitization effect mediated by VE-821. Altogether, our results demonstrate that 3D-cell architecture-associated resistance to cisplatin is due to an efficient induction of REV3L and TLS, dependent of ATR. Thus co-treatment with ATR inhibitors might be a promising strategy for enhancement of cisplatin treatment efficiency in breast cancer patients.

PIP4K2A and PIP4K2C transcript levels are associated with cytogenetic risk and survival outcomes in acute myeloid leukemia.

Phosphoinositide signaling pathway orchestrates primordial molecular and cellular functions in both healthy and pathologic conditions. Phosphatidylinositol-5-phosphate 4-kinase type 2 lipid kinase (PIP4K2) family, which compromises PIP4K2A, PIP4K2B and PIP4K2C, has drawn the attention in human cancers. Particularly in hematological malignancies, PIP4K2A was already described as an essential protein for a malignant phenotype, although the clinical and biological impact of PIP4K2B and PIP4K2C proteins have not being explored in the same extent. In the present study, we investigated the impact on clinical outcomes and gene network of PIP4K2A, PIP4K2B and PIP4K2C mRNA transcripts in acute myeloid leukemia (AML) patients included in The Cancer Genome Atlas (2013) study. Our results indicate that PIP4K2A and PIP4K2C, but not PIP4K2B, mRNA levels were significantly reduced in AML patients assigned to the favorable risk group (p < 0.05) and low levels of PIP4K2A and PIP4K2C positively affect clinical outcomes of AML patients (p < 0.05). Gene set enrichment analyses indicate that the expression of PIP4K2 genes is associated with biological process such as signal transduction, metabolism of RNA and genomic instability related-gene sets. In summary, our study provides additional evidence of the involvement of members of the PIP4K2 family, in particular PIP4K2A and PIP4K2C, in AML.

Interplay among steroids, body condition and immunity in response to long-term captivity in toads.

Stressful experiences can promote harmful effects on physiology and fitness. However, stress-mediated hormonal and immune changes are complex and may be highly dependent on body condition. Here, we investigated captivity-associated stress effects, over 7, 30, 60, and 90 days on plasma corticosterone (CORT) and testosterone (T) levels, body index, and innate immunity (bacterial killing ability and phagocytosis of peritoneal cells) in toads (Rhinella icterica). Toads in captivity exhibited elevated CORT and decreased T and immunity, without changes in body index. The inter-relationships between these variables were additionally contrasted with those obtained previously for R. schneideri, a related species that exhibited extreme loss of body mass under the same captive conditions. While T and phagocytosis were positively associated in both species, the relationship between CORT and bacterial killing ability was dependent on body index alterations. While CORT and bacterial killing ability were positively associated for toads that maintained body index, CORT was negatively associated with body index in toads that lost body mass over time in captivity. In these same toads, body index was positively associated with bacterial killing ability. These results demonstrate that steroids-immunity inter-relationships arising from prolonged exposure to a stressor in toads are highly dependent on body condition.

Expression of the Circadian Clock Gene BMAL1 Positively Correlates With Antitumor Immunity and Patient Survival in Metastatic Melanoma.

INTRODUCTION: Melanoma is the most lethal type of skin cancer, with increasing incidence and mortality rates worldwide. Multiple studies have demonstrated a link between cancer development/progression and circadian disruption; however, the complex role of tumor-autonomous molecular clocks remains poorly understood. With that in mind, we investigated the pathophysiological relevance of clock genes expression in metastatic melanoma. METHODS: We analyzed gene expression, somatic mutation, and clinical data from 340 metastatic melanomas from The Cancer Genome Atlas, as well as gene expression data from 234 normal skin samples from genotype-tissue expression. Findings were confirmed in independent datasets. RESULTS: In melanomas, the expression of most clock genes was remarkably reduced and displayed a disrupted pattern of co-expression compared to the normal skins, indicating a dysfunctional circadian clock. Importantly, we demonstrate that the expression of the clock gene aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) positively correlates with patient overall survival and with the expression of T-cell activity and exhaustion markers in the tumor bulk. Accordingly, high BMAL1 expression in pretreatment samples was significantly associated with clinical benefit from immune checkpoint inhibitors. The robust intratumoral T-cell infiltration/activation observed in patients with high BMAL1 expression was associated with a decreased expression of key DNA-repair enzymes, and with an increased mutational/neoantigen load. CONCLUSION: Overall, our data corroborate previous reports regarding the impact of BMAL1 expression on the cellular DNA-repair capacity and indicate that alterations in the tumor-autonomous molecular clock could influence the cellular composition of the surrounding microenvironment. Moreover, we revealed the potential of BMAL1 as a clinically relevant prognostic factor and biomarker for T-cell-based immunotherapies.

Non-Metastatic Cutaneous Melanoma Induces Chronodisruption in Central and Peripheral Circadian Clocks.

The biological clock has received increasing interest due to its key role in regulating body homeostasis in a time-dependent manner. Cancer development and progression has been linked to a disrupted molecular clock; however, in melanoma, the role of the biological clock is largely unknown. We investigated the effects of the tumor on its micro- (TME) and macro-environments (TMaE) in a non-metastatic melanoma model. C57BL/6J mice were inoculated with murine B16-F10 melanoma cells and 2 weeks later the animals were euthanized every 6 h during 24 h. The presence of a localized tumor significantly impaired the biological clock of tumor-adjacent skin and affected the oscillatory expression of genes involved in light- and thermo-reception, proliferation, melanogenesis, and DNA repair. The expression of tumor molecular clock was significantly reduced compared to healthy skin but still displayed an oscillatory profile. We were able to cluster the affected genes using a human database and distinguish between primary melanoma and healthy skin. The molecular clocks of lungs and liver (common sites of metastasis), and the suprachiasmatic nucleus (SCN) were significantly affected by tumor presence, leading to chronodisruption in each organ. Taken altogether, the presence of non-metastatic melanoma significantly impairs the organism's biological clocks. We suggest that the clock alterations found in TME and TMaE could impact development, progression, and metastasis of melanoma; thus, making the molecular clock an interesting pharmacological target.

Deletion and low expression of NFKBIA are associated with poor prognosis in lower-grade glioma patients.

Lower-grade gliomas (LGGs), which are uniformly fatal in young adults, are classified as grades II-III tumors according to their histological features. The NFκB transcription factor, a crucial player in cancer initiation and progression, is inactivated in the cytoplasm by inhibitory proteins (IκBs) that have been shown to exert tumor-suppressor activity. Therefore, using The Cancer Genome Atlas copy number alteration and RNA-Seq data from 398 patients, we evaluated the association between the expression and dosage of NFKBIA, which encodes IκBα, and the overall malignancy of LGGs. Deletion and low expression of NFKBIA were associated with enhanced tumor aggressiveness and poor prognosis in LGGs. Accordingly, the dosage and expression of NFKBIA were independent prognostic factors for 5-year survival (dosage: P = 0.016; expression: P = 0.002) and 5-year recurrence-free survival (dosage: P = 0.009; expression: P = 0.005). Moreover, gene set enrichment analyses and co-expression network analyses indicated a role for NFKBIA in the negative regulation of cell proliferation, possibly through the modulation of downstream NFκB activation. Overall, the present findings reveal the prognostic value of NFKBIA in LGGs, reinforcing the relevance of NFκB signaling in the development and progression of gliomas.