Co-culture Approach Research Articles

Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems.

Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability in vitro in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications. Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.

Endophytic Fungi Co-Culture: An Alternative Source of Antimicrobial Substances.

Antimicrobial resistance is becoming a critical issue due to the widespread and indiscriminate use of antibiotics and antifungals to treat common infections, leading to a growing shortage of effective drugs. Moreover, the increase in antimicrobial resistance is enhancing the pathogenicity and virulence of various pathogens. Microorganisms are key sources of chemically diverse specialized metabolites, which are produced in the final stages of their growth cycle. These metabolites hold significant value in chemical, pharmaceutical, and agrochemical industries. One of the major challenges researchers face in this field is the frequent isolation of already-known substances when classical protocols are used. To address this, several innovative strategies have been developed. The co-culture approach is a powerful tool for activating silent biosynthetic gene clusters, as it simulates natural microbial environments by creating artificial microbial communities. This method has shown promising results, with new compounds being isolated and the yields of target substances being improved. In this context, this review provides examples of antimicrobial compounds obtained from co-cultures of endophytic fungi, conducted in both liquid and solid media. Additionally, the review discusses the advantages and challenges of the co-culture technique. Significance and Impact of the Study: Microbial co-culture is a valuable strategy for discovering new natural products with antimicrobial activity, as well as for scaling up the production of target substances. This review aims to summarize important examples of endophyte co-cultures and highlights the potential of endophytic fungi co-culture for pharmacological applications.

Strategies for enhancing short- and medium-chain fatty acids synthesis from corn stover-based sugar: A new perspective on co-culture

In this study, corn stover-based sugar served as the initial carbon source to investigate the impact of different co-culture strategies on the synthesis of short- and medium-chain fatty acids (SMCFAs) through carbon chain elongation (CCE). The results indicated that the sequential inoculation outperformed simultaneous inoculation in the M. elsdenii and Lactobacillus groups. The inclusion of Lactobacillus significantly enhanced substrate utilization, generating lactate that facilitated the integration of odd carbon carboxylates (such as propionate and valerate) into the CCE products. The caproate concentration in the M. elsdenii+L. pentosus group reached 3.4 g/L, which was 1.3 times that in the control group. Furthermore, the introduction of C. kluyveri effectively boosted the synthesis of butyrate and caproate through CCE using ethanol as an electron donor within the co-culture setup of the M. elsdenii+C. kluyveri group. Moreover, the carbon efficiency of the co-culture group increased by 10–21 % compared to the control group, and the cell protein titer was 1.5–2 times higher than that of the control group. This study has established a method for co-producing SMCFAs and cell proteins utilizing bio-sugars through a co-culture approach, offering implications for lignocellulose utilization and potential high-value applications of SMCFAs in the future.

Bacillus methylotrophicus influences on the virulence of Aeromonas hydrophila and probiotic applications in Ctenopharyngodon idella

Probiotics are alternatives to antibiotics that resist infection and benefit the host in several aspects. Here, we isolated and identified a novel strain of Bacillus methylotrophicus (XA-8) with a broad spectrum of activity against Aeromonas hydrophila AhX040 and other aquatic bacterial pathogens. The strain was non-pathogenic as it revealed no cytotoxic effects in hepatic L8824 cells or pathophysiological signs in the tissue of Ctenopharyngodon idella (grass carp). Using a co-culture approach, XA-8 (1×109 CFU mL−1) when co-cultivated with AhX040 (1×108 CFU mL−1) revealed a reduction in the cell density, cytotoxicity, and infection rate of AhX040. More interestingly, genes such as aer, act, hylA, lafA, hcp, and luxS, associated with the virulence of AhX040, were significantly down-regulated during co-cultivation (P<0.05). Furthermore, XA-8 as a feed supplement to grass carp up-regulated the immune-related genes in the liver, spleen, kidney, and intestine. The villi length and expression level of growth-related genes in the intestine were significantly higher (P<0.05) in the XA-8 supplemented grass carp. In addition to resisting infection (52.63 %), probiotic XA-8 modulated the gut microbiota of grass carp by increasing Fusobacteriota and Bacteroidota at the phylum level, while decreasing Aeromonas at the genus level. The whole genome sequence analysis showed the strain possesses multiple gene clusters for antimicrobial substances and metabolism. Our results demonstrate that B.methylotrophicus XA-8 is a novel probiotic with potential applications for aquaculture.

Human Umbilical Cord-Mesenchymal Stem Cells Promote Extracellular Matrix Remodeling in Microglia.

Human mesenchymal stem cells modulate the immune response and are good candidates for cell therapy in neuroinflammatory brain disorders affecting both adult and premature infants. Recent evidence indicates that through their secretome, mesenchymal stem cells direct microglia, brain-resident immune cells, toward pro-regenerative functions, but the mechanisms underlying microglial phenotypic transition are still under investigation. Using an in vitro coculture approach combined with transcriptomic analysis, we identified the extracellular matrix as the most relevant pathway altered by the human mesenchymal stem cell secretome in the response of microglia to inflammatory cytokines. We confirmed extracellular matrix remodeling in microglia exposed to the mesenchymal stem cell secretome via immunofluorescence analysis of the matrix component fibronectin and the extracellular crosslinking enzyme transglutaminase-2. Furthermore, an analysis of hallmark microglial functions revealed that changes in the extracellular matrix enhance ruffle formation by microglia and cell motility. These findings point to extracellular matrix changes, associated plasma membrane remodeling, and enhanced microglial migration as novel mechanisms by which mesenchymal stem cells contribute to the pro-regenerative microglial transition.

In vitro co-culture system for investigating Armillaria root rot in Prunus spp. using a fiber-supported liquid approach.

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes ('Guardian®', 'MP-29', and Prunus cerasifera '14-4') were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp.

Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies.Key points• Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites• Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes• A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposedGraphical

Efficient butanol production from sweet sorghum stem juice by a co-culture system at an optimum temperature: Insights from oxidation-reduction potential monitoring and pilot scale validation

A two-stage co-culture approach was employed in an acetone-butanol-ethanol (ABE) fermentation. An obligate aerobic bacterium, Arthrobacter sp., was first grown for 6 h at 30 °C to create anaerobic conditions. Subsequently, Clostridium beijerinckii TISTR 1461 was inoculated and a fermentation was performed at 37 °C. To identify an intermediate temperature suitable for both microorganisms, their growth was examined at 30, 34, and 37 °C. C.beijerinckii exhibited the highest specific growth rate at 37 °C, while Arthrobacter sp. displayed similar growth rates at all tested temperatures. Butanol production from a synthetic medium (P2 medium) by C. beijerinckii at different temperatures using oxygen-free nitrogen (OFN) gas flushing as a control treatment revealed that fermentation at 37 °C gave the highest butanol concentration (PB, 9.98 g/L). Consequently, 37 °C was chosen for butanol production from sweet sorghum stem juice (SSJ) by co-culture of these two microorganisms in 1-L screw–capped bottles. Compared to the control treatment, higher PB (11.38 g/L), yield (YB/S, 0.37 g/g) and productivity (QB, 0.24 g/L·h) were achieved using the co-culture system. These results were further confirmed by monitoring the oxidation–reduction potential (ORP) during ABE fermentation in a 2-L stirred-tank bioreactor (STR). Moreover, when the co-culture fermentation at 37 °C was scaled up in a 30-L STR, the PB, YB/S and QB values were comparable to those obtained in the 2-L STR. Therefore, co-culture fermentation of Arthrobacter sp. and C.beijerinckii TISTR 1461 at 37 °C represents a promising method for large-scale butanol production.

Francisella tularensis Live Vaccine Strain training of murine alveolar and bone marrow-derived macrophages.

The discovery of non-specific "trained immunity" in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g., Mycobacterium bovis Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with Francisella tularensis Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium Listeria monocytogenes. The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous in vitro co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination.

Design and development of in-vitro co-culture device for studying cellular crosstalk in varied tissue microenvironment

Despite of being in different microenvironment, breast cancer cells influence the bone cells and persuade cancer metastasis from breast to bone. Multiple co-culture approaches have been explored to study paracrine signaling between these cells and to study the progression of cancer. However, lack of native tissue microenvironment remains a major bottleneck in existing co-culture technologies. Therefore, in the present study, a tumorigenic and an osteogenic microenvironment have been sutured together to create a multi-cellular environment and has been appraised to study cancer progression in bone tissue. The PCL-polystyrene and PCL-collagen fibrous scaffolds were characterized for tumorigenic and osteogenic potential induction on MDA-MB-231 and MC3T3-E1 cells respectively. Diffusion ability of crystal violet, glucose, and bovine serum albumin across the membrane were used to access the potential paracrine interaction facilitated by device. While in co-cultured condition, MDA-MB-231 cells showed EMT phenotype along with secretion of TNFα and PTHrP which lower down the expression of osteogenic markers including alkaline phosphatase, RUNX2, Osteocalcin and Osteoprotegerin. The cancer progression in bone microenvironment demonstrated the role and necessity of creating multiple tissue microenvironment and its contribution in studying multicellular disease progression and therapeutics.

IL-37 ameliorates myocardial fibrosis by regulating mtDNA-enriched vesicle release in diabetic cardiomyopathy mice

BackgroundDiabetic cardiomyopathy (DCM), a serious complication of diabetes, leads to structural and functional abnormalities of the heart and ultimately evolves to heart failure. IL-37 exerts a substantial influence on the regulation of inflammation and metabolism. Whether IL-37 is involved in DCM is unknown.MethodsThe plasma samples were collected from healthy controls, diabetic patients and DCM patients, and the level of IL-37 and its relationship with heart function were observed. The changes in cardiac function, myocardial fibrosis and mitochondrial injury in DCM mice with or without IL-37 intervention were investigated in vivo. By an in vitro co-culture approach involving HG challenge of cardiomyocytes and fibroblasts, the interaction carried out by cardiomyocytes on fibroblast profibrotic activation was studied. Finally, the possible interactive mediator between cardiomyocytes and fibroblasts was explored, and the intervention role of IL-37 and its relevant molecular mechanisms.ResultsWe showed that the level of plasma IL-37 in DCM patients was upregulated compared to that in healthy controls and diabetic patients. Both recombinant IL-37 administration or inducing IL-37 expression alleviated cardiac dysfunction and myocardial fibrosis in DCM mice. Mechanically, hyperglycemia impaired mitochondria through SIRT1/AMPK/PGC1α signaling, resulting in significant cardiomyocyte apoptosis and the release of extracellular vesicles containing mtDNA. Fibroblasts then engulfed these mtDNA-enriched vesicles, thereby activating TLR9 signaling and the cGAS-STING pathway to initiate pro-fibrotic process and adverse remodeling. However, the presence of IL-37 ameliorated mitochondrial injury by preserving the activity of SIRT1-AMPK-PGC1α axis, resulting in a reduction in release of mtDNA-enriched vesicle and ultimately attenuating the progression of DCM.ConclusionsCollectively, our study demonstrates a protective role of IL-37 in DCM, offering a promising therapeutic agent for this disease.Graphical abstractHyperglycemia aggravates mitochondrial injury through SIRT1/AMPK/PGC1α signaling, resulting in significant cardiomyocyte apoptosis and the release of extracellular vesicles containing mtDNA in DCM mice. Fibroblasts then engulf these mtDNA-enriched vesicles, activating TLR9 signaling and the cGAS-STING pathway to initiate profibrotic process and adverse remodeling. However, both exogenous and endogenous IL-37 ameliorate mitochondrial injury by preserving the activity of SIRT1-AMPK-PGC1α axis, and reducing the release of mtDNA-enriched vesicles, which attenuates the progression of DCM

OPFR removal by white rot fungi: screening of removers and approach to the removal mechanism.

The persistent presence of organophosphate flame retardants (OPFRs) in wastewater (WW) effluents raises significant environmental and health concerns, highlighting the limitations of conventional treatments for their remotion. Fungi, especially white rot fungi (WRF), offer a promising alternative for OPFR removal. This study sought to identify fungal candidates (from a selection of four WRF and two Ascomycota fungi) capable of effectively removing five frequently detected OPFRs in WW: tributyl phosphate (TnBP), tributoxy ethyl phosphate (TBEP), trichloroethyl phosphate (TCEP), trichloro propyl phosphate (TCPP) and triethyl phosphate (TEP). The objective was to develop a co-culture approach for WW treatment, while also addressing the utilization of less assimilable carbon sources present in WW. Research was conducted on carbon source uptake and OPFR removal by all fungal candidates, while the top degraders were analyzed for biomass sorption contribution. Additionally, the enzymatic systems involved in OPFR degradation were identified, along with toxicity of samples after fungal contact. Acetate (1.4 g·L-1), simulating less assimilable organic matter in the carbon source uptake study, was eliminated by all tested fungi in 4 days. However, during the initial screening where the removal of four OPFRs (excluding TCPP) was tested, WRF outperformed Ascomycota fungi. Ganoderma lucidum and Trametes versicolor removed over 90% of TnBP and TBEP within 4 days, with Pleorotus ostreatus and Pycnoporus sanguineus also displaying effective removal. TCEP removal was challenging, with only G. lucidum achieving partial removal (47%). A subsequent screening with selected WRF and the addition of TCPP revealed TCPP's greater susceptibility to degradation compared to TCEP, with T. versicolor exhibiting the highest removal efficiency (77%). This observation, plus the poor degradation of TEP by all fungal candidates suggests that polarity of an OPFR inversely correlates with its susceptibility to fungal degradation. Sorption studies confirmed the ability of top-performing fungi of each selected OPFR to predominantly degrade them. Enzymatic system tests identified the CYP450 intracellular system responsible for OPFR degradation, so reactions of hydroxylation, dealkylation and dehalogenation are possibly involved in the degradation pathway. Finally, toxicity tests revealed transformation products obtained by fungal degradation to be more toxic than the parent compounds, emphasizing the need to identify them and their toxicity contributions. Overall, this study provides valuable insights into OPFR degradation by WRF, with implications for future WW treatment using mixed consortia, emphasizing the importance of reducing generated toxicity.

Fibroblasts from HPV-negative oropharynx squamous cell carcinomas stimulate the release of osteopontin from cancer cells via the release of IL-6.

HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood. To investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV- OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk. A retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication. HPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV-positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV- OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs. This investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

Valorization of Cashew Apple Waste into a Low-Alcohol, Healthy Drink Using a Co-Culture of Cyberlindnera rhodanensis DK and Lactobacillus pentosus A14-6.

This study investigated the potential of microbial fermentative transforming processes in valorizing the cashew apple by-product into a low-alcohol, health-benefiting beverage. We particularly investigated the use of a non-Saccharomyces yeast, Cyberlindnera rhodanensis DK, as the main targeted microbe. At 30 °C without agitation, C. rhodanensis DK caused changes in key parameters during the fermentation of cashew apple juice (CAJ) in terms of varied pH values and initial sugar concentrations. This result indicated that pure CAJ, with pH adjusted to 6 and with the original 6.85% (w/v) total sugar content, was the most feasible condition, as glucose and fructose were mostly consumed at 12 days of fermentation. A co-culture approach with either Saccharomyces cerevisiae TISTR 5088 or Lactobacillus pentosus A14-6 was investigated to improve both physicochemical and fermentation characteristics. Co-fermentation with S. cerevisiae TISTR 5088 resulted in significantly increased ethanol accumulation to 33.61 ± 0.11 g/L, but diminished bioactive compounds, antioxidant activity, and antidiabetic potential. In contrast, co-fermentation with L. pentosus A14-6 demonstrated excellent outcomes, as it significantly increased sugar consumption and finally remained at only 4.95 g/L compared to C. rhodanensis DK alone, produced lower levels of ethanol at only 19.47 ± 0.06 g/L, and higher total titratable acid (TTA), resulting in a final pH of 3.6. In addition, co-fermentation with this lactic acid bacterium significantly enhanced bioactive compounds and antioxidant activity and also retained potential antidiabetic properties. These findings highlight the feasibility of using tailored microbial fermentation strategies to produce low-alcohol beverages with enhanced health-promoting properties from CAJ; however, product-development processes following health food regulations and sensory evaluation are necessary.

Co-culturing a multistrain Gram-negative inoculant useful in sustainable agriculture

Microbial-based inoculants for agricultural use consist of different strains. The consortia production process involves growing pure cultures separately and combining them in the appropriate ratio. However, the inclusion of multiple strains in the formulation increases production costs. By developing co-culture fermentations, it is possible to create consortia with the correct bacterial charge. This study aims to develop a cost-effective co-culture approach for producing an inoculum that includes the appropriate ratio of four Gram-negative bacteria, i.e., Azospirillum brasilense, Burkholderia ambifaria, Gluconacetobacter diazotrophicus, and Herbaspirillum seropedicae. The specific growth rates of strains were studied using the T4 medium, previously optimized for their culture. The co-fermentation process was optimized in 500 mL flasks to attain an equivalent density of 9.7-10 Log CFU mL-1. Then, it was successfully scaled up to a 5 L bioreactor, obtaining an equivalent density of 9.7-9.9 CFU mL-1. This first co-formulation of a four multistrain consortium formed by Gram-negative plant growth-promoting bacteria pave the road for future evaluations of other products useful for sustainable agriculture.

Abstract 1989: Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment

Abstract Introduction: Non-small cell lung cancers (NSCLC) harboring EGFR mutations are commonly treated with EGFR tyrosine kinase inhibitors (TKIs), but the emergence of drug resistance often leads to treatment failure. While various acquired resistance mechanisms have been identified, they are not always actionable. Furthermore, since drug resistance is typically studied after cancer relapse, little is known about the cellular or microenvironmental taking place during the formation of resistance at the stage of minimal residual disease (MRD). Therefore, it is necessary to understand the mechanisms leading to resistance during MRD and develop new ways to prevent drug resistance in EGFR-mutant cancers. Methods: In this study, we discovered pre-existing intratumor heterogeneity in EGFR expression within EGFR-mutant NSCLCs and found that patient tumors contain clones with low mutant EGFR expression. To study the effect of this intratumor heterogeneity on drug response, we isolated low and high expressing cells and conducted longitudinal drug challenge assays in ex vivo and in vivo models. To dissect how the observed heterogeneity affects drug resistance and the tumor microenvironment during MRD, we employed patient-derived tumor cells and used novel tumor-on-a-chip co-culture approaches. Results: Our findings reveal that EGFR-low cells are more tolerant to drug treatment and can survive the initial TKI insult, making them more likely to contribute to drug resistance in EGFR-mutant NSCLCs. Additionally, the surviving EGFR-low cells were able to modify the tumor microenvironment leading to fibroblast accumulation and immune suppression, contributing to the drug-tolerant tumor microenvironment. Importantly, we found that pharmacological induction of EGFR using epigenetic inhibitors was able to sensitize the drug tolerant cells to EGFR inhibition, suggesting a potential strategy to overcome TKI resistance in NSCLC patients. Conclusions: In conclusion, our study sheds light on the critical role of intratumoral heterogeneity in treatment resistance and offers valuable insights into potential strategies to overcome this challenge. Our findings suggest that combination therapies that can alter the cellular phenotype during MRD have the potential to prevent intrinsic drug resistance. This underscores the importance of gaining a better understanding of the mechanisms underlying drug-tolerant microenvironment and developing more effective treatment strategies for patients with EGFR-mutant NSCLCs. Citation Format: Heidi M. Haikala, Bassel Alsaed, Jieun Son, Linh Lin, Prafulla Gokhale, Pasi Jänne. Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1989.

Co-culturing of Saccharomycopsis fibuligera and Lacticaseibacillus paracasei to improve the bioactive components and flavor characteristics of Dendrobium officinale

In this study, Saccharomycopsis fibuligera FBKL2.8DCJS1 and Lacticaseibacillus paracasei FBKL1.3028 were jointly introduced into Dendrobium officinale liquid to probe alterations in their active constituents. Over a 15-day fermentation period, analyses were conducted to ascertain shifts in physicochemical attributes, antioxidant capacity, metabolite transformation, flavor profiles, and sensory characteristics. The findings revealed augmented color differentiation, increased antioxidant potential, and heightened acidity post-fermentation. This finding points to a positive correlation between acidity and color variation and antioxidant capacity. The microbial fermentation process was advantageous for ameliorating both the color and taste of D. officinale while also bolstering its antioxidant capacity. Additionally, several bioactive substances favorable to the organism, including metabolites such as proscillaridin, D-sorbitol, and glucomannan, were found to be upregulated postfermentation. The most enriched metabolic pathways identified in the KEGG metabolic pathway were purine metabolism and amino acid metabolism. Notably, co-cultivating S. fibuligera and L. paracasei amplified the production of ethyl lactate, indicating that this co-culture approach induced higher levels of this compound. Consequently, fermenting D. officinale with S. fibuligera and L. paracasei not only enhanced its bioactive properties but also enhanced its flavor profile.

Enhancing Beverage Fermentation through Synergy of Saccharomycopsis fibuligera and Saccharomyces cerevisiae: A Mini-Review

Microbial fermentation; a natural process dating back over 7000 years BC, plays a pivotal role in beverage production. While Saccharomyces cerevisiae dominates the industry, recent research emphasizes the importance of co-culture with non-Saccharomyces yeasts for enhanced flavor and aroma. This review explores the cooperative interaction between Saccharomycopsis fibuligera and S. cerevisiae in alcoholic fermentation, shedding light on their enzymatic capabilities. S. fibuligera, an ascomycete with potent amylolytic activity, demonstrates the ability to efficiently convert starch into alcohol, contributing to improved fermentation stability. Co-culturing with S. cerevisiae unleashes a biochemical diversity that enhances the sensory attributes of beverages. Beyond flavor complexity, the co-culture strategy influences key compounds, including phenolic compounds and esters, elevating overall quality. The review delves into the biochemical intricacies of starch-based fermentation, emphasizing the potential of S. fibuligera in hydrolyzing starch into fermentable sugars. S. cerevisiae, a versatile and genetically diverse yeast, adapts to different environmental conditions crucial for successful fermentation. The co-culture approach not only accelerates fermentation but also combats contamination and reduces overall processing time.

Photo-addressable microwell devices for rapid functional screening and isolation of pathogen inhibitors from bacterial strain libraries.

Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.

Symbiodiniaceae photophysiology and stress resilience is enhanced by microbial associations

Symbiodiniaceae form associations with extra- and intracellular bacterial symbionts, both in culture and in symbiosis with corals. Bacterial associates can regulate Symbiodiniaceae fitness in terms of growth, calcification and photophysiology. However, the influence of these bacteria on interactive stressors, such as temperature and light, which are known to influence Symbiodiniaceae physiology, remains unclear. Here, we examined the photophysiological response of two Symbiodiniaceae species (Symbiodinium microadriaticum and Breviolum minutum) cultured under acute temperature and light stress with specific bacterial partners from their microbiome (Labrenzia (Roseibium) alexandrii, Marinobacter adhaerens or Muricauda aquimarina). Overall, bacterial presence positively impacted Symbiodiniaceae core photosynthetic health (photosystem II [PSII] quantum yield) and photoprotective capacity (non-photochemical quenching; NPQ) compared to cultures with all extracellular bacteria removed, although specific benefits were variable across Symbiodiniaceae genera and growth phase. Symbiodiniaceae co-cultured with M. aquimarina displayed an inverse NPQ response under high temperatures and light, and those with L. alexandrii demonstrated a lowered threshold for induction of NPQ, potentially through the provision of antioxidant compounds such as zeaxanthin (produced by Muricauda spp.) and dimethylsulfoniopropionate (DMSP; produced by this strain of L. alexandrii). Our co-culture approach empirically demonstrates the benefits bacteria can deliver to Symbiodiniaceae photochemical performance, providing evidence that bacterial associates can play important functional roles for Symbiodiniaceae.