Cassava (
Manihot esculenta
Crantz) is an important root crop, which despite its drought tolerance suffers considerable yield losses under water deficit. One strategy to increase crop yields under ...water deficit is improving the crop’s transpiration efficiency, which could be achieved by variety selection and potassium application. We assessed carbon isotope composition in bulk leaf material and extracted carbohydrates (soluble sugar, starch, and cellulose) of selected leaves one month after inducing water deficit to estimate transpiration efficiency and storage root biomass under varying conditions in a greenhouse experiment. A local and improved variety were grown in sand, supplied with nutrient solution with two potassium levels (1.44 vs. 0.04 mM K
+
) and were subjected to water deficit five months after planting. Potassium application and selection of the improved variety both increased transpiration efficiency of the roots with 58% and 85% respectively. Only in the improved variety were
13
C ratios affected by potassium application (up to - 1.8‰ in δ
13
C of soluble sugar) and water deficit (up to + 0.6‰ in δ
13
C of starch and soluble sugar). These data revealed a shift in substrate away from transitory starch for cellulose synthesis in young leaves of the improved variety under potassium deficit. Bulk δ
13
C of leaves that had fully developed prior to water deficit were the best proxies for storage root biomass (r = - 0.62, r = - 0.70) and transpiration efficiency (r = - 0.68, r = - 0.58) for the local and improved variety respectively, making laborious extractions redundant. Results obtained from the youngest fully developed leaf, commonly used as a diagnostic leaf, were complicated by remobilized assimilates in the improved variety, making them less suitable for carbon isotope analysis. This study highlights the potential of carbon isotope composition to assess transpiration efficiency and yield, depending on the chosen sampling strategy as well as to unravel carbon allocation processes.
Rationale
Cassava production faces challenges in a changing climate. Pulse labelling cassava with 13C‐CO2 has the potential to elucidate carbon allocation mechanisms of cassava under drought stress ...and with potassium application. Understanding these mechanisms could guide efforts to mitigate effects of drought in cassava cropping systems.
Methods
Forty‐eight cassava plants received a nutrient solution high or low in potassium. Water deficit was imposed on half of the plants at bulk root initiation stage, after which they were labelled for 8 h with 13C‐CO2 in a 15 m3 growth chamber. Plants were harvested 8 h, 9 days and 24 days after labelling, and separated into leaves, stems and roots. δ13C values of the different parts were measured using an isotope ratio mass spectrometer, from which 13C excess was calculated.
Results
Water deficit decreased transpiration (P < 0.001) and increased carbon respiration (P < 0.05). Potassium application increased assimilate distribution to the roots (P < 0.05) at 9 days after labelling, more strongly for plants under water deficit. The opposite was found at 24 days (P < 0.05) with the legacy of water deficit additionally increasing assimilate distribution to roots (P < 0.05). Youngest, fully expanded leaves contained up to 47% of initial 13C excess at 24 days after labelling.
Conclusions
Pulse labelling proved to be successful in shedding light on carbon allocation in relation to water and potassium availability. This technique, once adapted to field conditions, could further be used to improve fertilizer recommendations or change agronomic practices to cope with plant stress.
Cassava production faces challenges in a changing climate. Pulse labelling cassava with
C-CO
has the potential to elucidate carbon allocation mechanisms of cassava under drought stress and with ...potassium application. Understanding these mechanisms could guide efforts to mitigate effects of drought in cassava cropping systems.
Forty-eight cassava plants received a nutrient solution high or low in potassium. Water deficit was imposed on half of the plants at bulk root initiation stage, after which they were labelled for 8 h with
C-CO
in a 15 m
growth chamber. Plants were harvested 8 h, 9 days and 24 days after labelling, and separated into leaves, stems and roots. δ
C values of the different parts were measured using an isotope ratio mass spectrometer, from which
C excess was calculated.
Water deficit decreased transpiration (P < 0.001) and increased carbon respiration (P < 0.05). Potassium application increased assimilate distribution to the roots (P < 0.05) at 9 days after labelling, more strongly for plants under water deficit. The opposite was found at 24 days (P < 0.05) with the legacy of water deficit additionally increasing assimilate distribution to roots (P < 0.05). Youngest, fully expanded leaves contained up to 47% of initial
C excess at 24 days after labelling.
Pulse labelling proved to be successful in shedding light on carbon allocation in relation to water and potassium availability. This technique, once adapted to field conditions, could further be used to improve fertilizer recommendations or change agronomic practices to cope with plant stress.
It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal ...adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma.
RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70
tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs.
In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70
tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70
tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner.
We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70
cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.
Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ...ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1).
Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling.
Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α
T-cell infiltration and activation in primary tumor and lymphoid organs.
Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.
Murine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a ...fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.
Intraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.
Mice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.
The current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.
The transition of ductal carcinoma
(DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic ...testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models.
Usutu virus (USUV) is a neurotropic flavivirus closely related to West Nile virus (WNV). Its enzootic cycle mainly involves mosquitoes and birds. Human infection can occur with occasional, but ...sometimes severe, neurological complications. Since its emergence and spread in Europe over the last two decades, USUV has been linked to significant avian outbreaks, especially among
, including European blackbirds (
). Strikingly, no in vivo avian model exists so far to study this arbovirus. The domestic canary (
) is a passerine, which is considered as a highly susceptible model of infection by WNV. Here, we experimentally challenged domestic canaries with two different doses of USUV. All inoculated birds presented detectable amounts of viral RNA in the blood and RNA shedding via feathers and droppings during the early stages of the infection, as determined by RT-qPCR. Mortality occurred in both infected groups (1/5 and 2/5, respectively) and was not necessarily correlated to a pure neurological disease. Subsequent analyses of samples from dead birds showed histopathological changes and virus tropism mimicking those reported in naturally infected birds. A robust seroconversion followed the infection in almost all the surviving canaries. Altogether, these results demonstrate that domestic canaries constitute an interesting experimental model for the study of USUV pathogenesis and transmission.
Early clinical results of two tau tracers, 18FT808 and 18FT807, have recently been reported. In the present study, the biodistribution, radiometabolite quantification, and competition-binding studies ...were performed in order to acquire comparative preclinical data as well as to establish the value of T808 and T807 as benchmark compounds for assessment of binding affinities of eight new/other tau tracers. Biodistribution studies in mice showed high brain uptake and fast washout. In vivo radiometabolite analysis using high-performance liquid chromatography showed the presence of polar radiometabolites in plasma and brain. No specific binding of 18FT808 was found in transgenic mice expressing mutant human P301L tau. In semiquantitative autoradiography studies on human Alzheimer disease slices, we observed more than 50% tau selective blocking of 18FT808 in the presence of 1 µmol/L of the novel ligands. This study provides a straightforward comparison of the binding affinity and selectivity for tau of the reported radiolabeled tracers BF-158, BF-170, THK5105, lansoprazole, astemizole, and novel tau positron emission tomography ligands against T807 and T808. Therefore, these data are helpful to identify structural requirements for selective interaction with tau and to compare the performance of new highly selective and specific radiolabeled tau tracers.
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