Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Neutrophil-to-lymphocyte ratio is a prognostic factor reflecting immune condition of tumor microenvironment in squamous cell lung cancer.

Inflammatory factors in the peripheral blood, such as the C-reactive protein level and neutrophil-to-lymphocyte ratio (NLR), are prognostic markers in multiple types of cancer, including non-small cell lung cancer (NSCLC). However, the association between inflammatory factors and prognosis based on histological types has not been adequately reported. In addition, the relationship between these factors and the immune condition of the tumor microenvironment (TME) is unclear. In this single center, retrospective study, we first investigated the relationship between preoperative inflammatory markers and clinical outcomes in 176 patients with NSCLC who underwent surgery. Lung adenocarcinoma (LUAD) showed no significant prognostic marker, whereas for lung squamous cell carcinoma (LUSC), a multivariate analysis showed that a high NLR was significantly associated with postoperative recurrence. In LUSC patients, the median time of postoperative recurrence-free survival in patients with a low NLR was longer than that in patients with a high NLR. We then compared the tumor-infiltrating lymphocyte (TIL) profile with inflammatory markers in peripheral blood and found that the NLR was negatively correlated with the frequencies of T cells and B cells in LUSC tissues. Thus, the NLR is a useful predictive biomarker for postoperative recurrence and may reflect the immune condition of the TME in LUSC.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

EVI1 exerts distinct roles in AML via ERG and cyclin D1 promoting a chemoresistant and immune-suppressive environment.

Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1-transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation-related short hairpin RNAs revealed that the erythroblast transformation-specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer-cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

Severe autoimmune pancytopenia after autologous hematopoietic stem cell transplantation for Hodgkin lymphoma.

Autoimmune pancytopenia is rarely seen with Hodgkin lymphoma, and only one pediatric case of pancytopenia after autologous hematopoietic stem cell transplantation (HSCT) has been reported. We herein report a case of autoimmune pancytopenia that developed after autologous HSCT for nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). A 56-year-old Japanese woman underwent autologous HSCT for NLPHL. She developed autoimmune pancytopenia seven months after autologous HSCT. In this case, PSL was effective, and the blood cell counts normalized completely. However, the patient suffered from a fatal infection, probably because of immunosuppression caused by prolonged administration of PSL, as well as a history of several chemotherapies and autologous HSCT. To our knowledge, this is the first adult case of autoimmune pancytopenia after autologous HSCT for Hodgkin lymphoma. To further validate the optimal treatment strategy for autoimmune cytopenia after autologous HSCT, more cases are necessary.

Heterozygous Dnmt3a R878C induces expansion of quiescent hematopoietic stem cell pool.

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

CAMK2G is identified as a novel therapeutic target for myelofibrosis.

Although JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

A case of thymoma showing significant tumor reduction after anti-thymocyte globulin.

A 71-year-old female with type B3 thymoma developed severe aplastic anemia. Anti-thymocyte globulin was administered with glucocorticoids and cyclosporin A as the treatment for aplastic anemia. Computed tomography scan revealed that thymoma apparently shrank and remained without regrowth for at least 7 months. As previously reported, glucocorticoid has therapeutic effects on thymoma especially with abundant lymphocytes. Anti-thymocyte globulin also depletes peripheral lymphocytes, but its efficacy in the treatment of thymoma is unknown. Anti-thymocyte globulin and glucocorticoids may have cooperated with each other in reducing thymoma in our case. More cases should be accumulated to elucidate the effects of anti-thymocyte globulin on thymoma.

Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation.

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1-driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1-overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA-mediated knockdown selectively decreased Evi1-driven leukemogenesis in vitro. Moreover, pharmacological or shRNA-mediated Fbp1 inhibition in secondarily transplanted Evi1-overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.

Autophosphorylation of EGFR at Y954 Facilitated Homodimerization and Enhanced Downstream Signals.

Asymmetric dimer formation of epidermal growth factor receptor (EGFR) is crucial for EGF-induced receptor activation. Even though autophosphorylation is important for activation, its role remains elusive in the context of regulating dimers. In this study, employing overlapping time series analysis to raster image correlation spectroscopy (RICS), we observed time-dependent transient dynamics of EGFR dimerization and found EGFR kinase activity to be essential for dimerization. As a result of which, we hypothesized that phosphorylation could influence dimerization. Evaluating this point, we observed that one of the tyrosine residues (Y954) located in the C-terminal lobe of the activator kinase domain was important to potentiate dimerization. Functional imaging to monitor Ca2+ and ERK signals revealed a significant role of Y954 in influencing downstream signaling cascade. Crucial for stabilization of EGFR asymmetric dimer is a "latch" formed between kinase domains of the binding partners. Because Y954 is positioned adjacent to the latch binding region on the kinase domain, we propose that phosphorylation strengthened the latch interaction. On the contrary, we identified that threonine phosphorylation (T669) in the latch domain negatively regulated EGFR dimerization and the downstream signals. Overall, we have delineated the previously anonymous role of phosphorylation at the latch interface of kinase domains in regulating EGFR dimerization.

Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.

Computed tomography findings of early-stage TAFRO syndrome and associated adrenal abnormalities.

OBJECTIVES: To compare CT findings of early (within 3 weeks post-onset)- and later (within 1 month before or after diagnostic criteria were satisfied, and later than 3 weeks post-onset) stage thrombocytopenia, anasarca, fever, reticulin fibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome. METHODS: Between 2014 and 2019, 13 patients with TAFRO syndrome (8 men and 5 women; mean age, 54.9 years) from nine hospitals were enrolled. The number of the following CT findings (CT factors) was recorded: the presence of anasarca, organomegaly, adrenal ischaemia, anterior mediastinal lesion, bony lesion, and lymphadenopathy. Records of adrenal disorders (adrenomegaly, ischaemia, and haemorrhage) throughout the disease course were also collected. Differences in CT factors at each stage were statistically compared between remission and deceased groups. RESULTS: Para-aortic oedema and mild lymphadenopathy were observed in all patients, whereas pleural effusion, ascites, and subcutaneous oedema were found in 5/13, 7/13, and 7/13 cases, respectively, at the early stage. CT factors at the early stage were significantly higher in the deceased than in the remission group (mean, 11 vs 6.5; p = 0.04), while they were nonsignificant at the later stage. Adrenal disorders were present in 7/13 cases throughout the course including 6 of adrenomegaly and 4 of ischaemia at the early stage. CONCLUSIONS: Para-aortic oedema and mild lymphadenopathy are most common at the early stage. Anasarca, organomegaly, lymphadenopathy, and adrenal disorders on early-stage CT are useful for unfavourable prognosis prediction. Moreover, adrenal disorders are frequent even at the early stage and are useful for early diagnosis of TAFRO syndrome. KEY POINTS: • CT findings facilitate early diagnosis and prognosis prediction in TAFRO syndrome. • Adrenal disorders are frequently observed in TAFRO syndrome. • Adrenal disorders are useful for differential diagnosis of TAFRO syndrome.

Anti-GPRC5D/CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma.

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Polymeric Engineering of Nanoparticles for Highly Efficient Multifunctional Drug Delivery Systems.

Most targeting strategies of anticancer drug delivery systems (DDSs) rely on the surface functionalization of nanocarriers with specific ligands, which trigger the internalization in cancer cells via receptor-mediated endocytosis. The endocytosis implies the entrapment of DDSs in acidic vesicles (endosomes and lysosomes) and their eventual ejection by exocytosis. This process, intrinsic to eukaryotic cells, is one of the main drawbacks of DDSs because it reduces the drug bioavailability in the intracellular environment. The escape of DDSs from the acidic vesicles is, therefore, crucial to enhance the therapeutic performance at low drug dose. To this end, we developed a multifunctionalized DDS that combines high specificity towards cancer cells with endosomal escape capabilities. Doxorubicin-loaded mesoporous silica nanoparticles were functionalized with polyethylenimine, a polymer commonly used to induce endosomal rupture, and hyaluronic acid, which binds to CD44 receptors, overexpressed in cancer cells. We show irrefutable proof that the developed DDS can escape the endosomal pathway upon polymeric functionalization. Interestingly, the combination of the two polymers resulted in higher endosomal escape efficiency than the polyethylenimine coating alone. Hyaluronic acid additionally provides the system with cancer targeting capability and enzymatically controlled drug release. Thanks to this multifunctionality, the engineered DDS had cytotoxicity comparable to the pure drug whilst displaying high specificity towards cancer cells. The polymeric engineering here developed enhances the performance of DDS at low drug dose, holding great potential for anticancer therapeutic applications.

Amphiphilic Nanoaggregates with Bimodal MRI and Optical Properties Exhibiting Magnetic Field Dependent Switching from Positive to Negative Contrast Enhancement.

Mixed micelles based on amphiphilic gadolinium(III)-DOTA and europium(III)-DTPA complexes were synthesized and evaluated for their paramagnetic and optical properties as potential bimodal contrast agents. Amphiphilic folate molecule for targeting the folate receptor protein, which is commonly expressed on the surface of many human cancer cells, was used in the self-assembly process in order to create nanoaggregates with targeting properties. Both targeted and nontargeted nanoaggregates formed monodisperse micelles having distribution maxima of 10 nm. The micelles show characteristic europium(III) emission with quantum yields of 2% and 1.1% for the nontargeted and targeted micelles, respectively. Fluorescence microscopy using excitation at 405 nm and emission at 575-675 nm was employed to visualize the nanoaggregates in cultured HeLa cells. The uptake of folate-targeted and nontargeted micelles is already visible after 5 h of incubation and was characterized with the europium(III) emission, which is clearly observable in the cytoplasm of the cells. The very fast longitudinal relaxivity r1 of ca. 26 s-1 mM-1 per gadolinium(III) ion was observed for both micelles at 60 MHz and 310 K. Upon increasing the magnetic field to 300 MHz, the nanoaggregates exhibited a large switching to transversal relaxivity with r2 value of ca. 52 s-1 mM-1 at 310 K. Theoretical fitting of the 1H NMRD profiles indicate that the efficient T1 and T2 relaxations are sustained by the favorable magnetic and electron-configuration properties of the gadolinium(III) ion, rotational correlation time, and coordinated water molecule. These nanoaggregates could have versatile application as a positive contrast agent at the currently used magnetic imaging field strengths and a negative contrast agent in higher field applications, while at the same time offering the possibility for the loading of hydrophobic therapeutics or targeting molecules.

Mapping Transient Protein Interactions at the Nanoscale in Living Mammalian Cells.

Protein-protein interactions (PPIs) form the basis of cellular processes, regulating cell behavior and fate. PPIs can be extremely transient in nature, which hinders their detection. In addition, traditional biochemical methods provided limited information on the spatial distribution and temporal dynamics of PPIs that is crucial for their regulation in the crowded cellular environment. Given the pivotal role of membrane micro- and nanodomains in the regulation of PPIs at the plasma membrane, the development of methods to visualize PPIs with a high spatial resolution is imperative. Here, we present a super-resolution fluorescence microscopy technique that can detect and map short-lived transient protein-protein interactions on a nanometer scale in the cellular environment. This imaging method is based on single-molecule fluorescence microscopy and exploits the effect of the difference in the mobility between cytosolic and membrane-bound proteins in the recorded fluorescence signals. After the development of the proof of concept using a model system based on membrane-bound modular protein domains and fluorescently labeled peptides, we applied this imaging approach to investigate the interactions of cytosolic proteins involved in the epidermal growth factor signaling pathway (namely, Grb2, c-Raf, and PLCγ1). The detected clusters of Grb2 and c-Raf were correlated with the distribution of the receptor at the plasma membrane. Additionally, the interactions of wild type PLCγ1 were compared with those detected with truncated mutants, which provided important information regarding the role played by specific domains in the interaction with the membrane. The results presented here demonstrate the potential of this technique to unravel the role of membrane heterogeneity in the spatiotemporal regulation of cell signaling.

Chronic myelogenous leukemia presenting with central nervous system infiltration, successfully treated with central nervous system-directed chemotherapy followed by allogeneic stem cell transplantation.

With the introduction of tyrosine kinase inhibitors (TKIs), prognosis of chronic myelogenous leukemia (CML) has improved dramatically. However, treatment for blast phase (BP) CML remains a challenge. CML infiltration of the central nervous system (CNS) is particularly rare and no effective treatment strategy has been established. The present case reports a 30-year-old man presenting with sensory deafness. Marked leukocytosis with p210 BCR-ABL1 mRNA positivity and Philadelphia chromosome detected by bone marrow biopsy confirmed the diagnosis of CML. Dura thickening in brain MRI and immature cells with Philadelphia chromosome in spinal fluid confirmed CNS invasion of CML and he was diagnosed with BP-CML. Two cycles of hyper-CVAD/MA (cyclophosphamide, vincristine, doxorubicin and dexamethasone/ high-dose methotrexate and cytarabine) therapy with dasatinib and concomitant intrathecal chemotherapy induced complete cytogenetic response and remission of CNS involvement. Bone marrow transplantation from an unrelated HLA-mismatched donor was performed and complete molecular response in bone marrow and complete remission in CNS disease was achieved. To our knowledge, this the first report of BP-CML with CNS infiltration at initial diagnosis, and shows that CNS-directed chemotherapy with dasatinib followed by allogeneic hematopoietic stem cell transplantation is useful in the treatment for BP-CML with CNS invasion in the TKI era.

Introducing fluorescence resonance energy transfer-based biosensors for the analysis of cAMP-PKA signalling in the fungal pathogen Candida glabrata.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP-PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP-PKA activity in this pathogen, we here present the usage of two FRET-based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time-resolved manner, as we exemplify by glucose-induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP-PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.