NPM1 mutated AMLs, which expressed B7-H3. These different B7 immune profiles suggest that specific immunotherapies are required to target the distinct immune evasion strategies of this genetically heterogeneous disease.Acute myeloid leukemia (AML) is generally considered a poorly immunogenic malignancy, displaying a “non-inflamed” leukemia microenvironment (LME), leading to T cell tolerance. However, the immune landscape of AML is much more heterogeneous. Since B7 expression is regarded as a consequence of an interferon-mediated “inflammatory” phenotype, we have investigated by flow cytometry the B7 checkpoint ligands B7.1, B7.2, programmed death ligand 1 (PD-L1), PD-L2, ICOS-L, B7-H3, and B7-H4 on the AML blasts of 30 newly diagnosed patients and their corresponding receptors  on bone marrow (BM) T cell maturation populations. We could thus evidence B7-negative and B7-positive leukemias either with an isolated expression or part of eight different checkpoint ligand “signatures” that always included an inhibitory B7 molecule. B7-positive AMLs encompassed intermediate and adverse European Leukemia Net (ELN) risk cases and displayed mainly central memory CD4+ T cells with high ICOS levels and effector CD8+ T cells with high PD-1 expression. B7-negative cases were rather classified as AML with recurrent genetic anomalies and displayed predominantly naive T cells, with the exception of  The last decade has witnessed dramatic advances in the field of cancer immunotherapy. Immune checkpoint blockade (ICB) is reshaping the treatment paradigm of solid tumors  and hemaSimultaneously, gene expression profiling (GEP) of tumor immune microenvironments is revolutionizing our understanding of cancer-immune interactions. Several recurrent pan-cancer immune profiles have been identified and could serve as biomarkers for predicting clinical responses to immunotherapy or for tailoring personalized treatment strategies , 8. BrieThe development of immunotherapy in acute myeloid leukemia (AML) has been hindered so far not only by its remarkable genetic, antigenic, and clonal heterogeneity –13 and tNoteworthy, the LME of some AML cases displays evidence of a prior antileukemic immune response, restrained by immune escape mechanisms such as cytotoxic T lymphocyte associated protein-4 (CTLA-4)/B7.1/B7.2 and programmed death 1 (PD-1)/PD-1L signaling or Treg expansion . B7 moleThe expression of IFN-γ-responsive genes has been correlated with primary refractory AML and is able to predict responses to ICB or flotetuzumab (CD3/CD123 DART®), showing that immune signatures within the LME can serve as reliable biomarkers to predict responses to immunotherapy , 29.Previous research was rather focused on finding prognostic relevance of isolated B7 molecule expression in AML , 30–32, Well-beyond investigating the expression of isolated molecules, our study aims to simultaneously evaluate the B7 checkpoint ligand phenotype of AML blasts  and the expression of immune checkpoint receptors (ICRs)  on helper and cytotoxic T cell maturation populations and to correlate these data to standard prognostic factors. We advanced the notion of “B7 checkpoint ligand signatures” to systematize the co-signaling output of AML blasts toward T cells. Since the expression of the immune escape PD-1/PD-L1 axis is correlated with an IFN-rich “inflamed” LME , it willde novo, non-promyelocytic AML between 2016 and 2019 at the Iaşi Regional Oncology Institute, Romania, were included in this study. This study has been approved by the institutional ethics committee. BM and peripheral blood (PB) samples were collected at diagnosis. AML diagnosis was established according to the WHO diagnostic criteria  on erythrocyte-lysed fresh BM and PB samples. This study comprised four phases:  confirmaData acquisition was performed on a BD FACS ARIA III cytometer, and data were interpreted using the FACS DIVA v6.1.3 software. An identical investigation protocol was applied for all healthy subjects. The MoAbs used in this study are detailed in AML blasts gating was performed on CD45+/CD34+/CD117+/HLA-DR+ events. Subsequently, the expression level of each B7 molecule was assessed.T cell gating was performed on CD3+/CD4+ and CD3+/CD8+ events. The following T cell maturation subsets were defined based on their differential expression of CD28, CD27, and CD45RA: naive (N), central memory (CM), intermediate effector memory (iEM), late effector memory (late EM) , 39. Tren, total number of patients, the significance p-value, the statistical test used. The chi square, Fisher's exact test, Mann–Whitney test, and Student's t-test were used to analyze the associations between different variables. The Pearson correlation coefficient was calculated to investigate the relationships between numerical variables. The two-way ANOVA test was used to analyze the differences among multiple variables. A p < 0.05 was considered as statistically significant.Statistical analyses were performed using the IBM® SPSS Statistics 21.0 Software. Each figure contains the relevant statistical information: the FLT-3-ITD mutations, and three patients had NPM1 mutated status. FLT-3-ITD and NPM-1 mutations did not coexist in our study group.Patient data are summarized in p = 0.028, B7– vs. HD: p = 0.739), B7.2  and ICOS-L . Out of the B7-positive cases, 10 expressed B7 molecule signatures and eight had isolated B7 expression.Within the healthy donor (HD) group, we could evidence the isolated expression of two molecules, PD-L1 and B7.2, in two cases , while 1n = 15), followed by PD-L1 , PD-L2 , ICOS-L , B7-H3 , and B7-H4 . B7.1 was expressed at extremely low levels and was thus considered negative. B7.2 was equally expressed isolated or co-expressed, while all the other B7 ligands were mainly co-expressed on AML blasts as B7 signatures.B7.2 was the most frequently expressed molecule  had intermediate or adverse ELN risk AML-Not Otherwise Specified (NOS). Out of these, complex karyotype AML-NOS expressed either B7.2 isolated or co-expressed B7.2, ICOS-L, and PD-L1, while normal karyotype AML-NOS also expressed PD-L2, B7-H3, and B7-H4 alongside B7.2, ICOS-L, and PD-L1 . The twopression .NPM1 mutated AML was the only B7-positive subtype and was correlated with B7-H3 expression (p = 0.02) and significantly higher levels of B7-H3 when compared to the AML-NOS cases (p = 0.019). AML with t  was B7 negative and expressed significantly lower percentages of B7.2 (p = 0.036) when compared to the AML-NOS cases . Out of this group, OS cases .p = 0.017, chi square test) and worse overall survival  (data not shown).Further details regarding the expression of each B7 checkpoint ligand relative to age, WHO AML type, and ELN risk are provided in We have identified eight different B7 checkpoint ligand signatures in 10 patients : co-exprp = 0.0002), B7.2–ICOS-L (p = 0.019), PD-L1–ICOS-L (p = 0.0009), PD-L1–B7-H4 (p = 0.0051), PD-L2–B7-H4 (p < 0.0001). B7.2 expression was rather associated with ICOS-L and PD-L1 in the B7.2/PD-L1/ICOS-L (three cases) and B7.2/PD-L1/ICOS-L/B7-H4 signatures (one case). Moreover, PD-L2 was correlated to B7-H4 expression (p < 0.0001). However, B7.2 expression was not correlated to PD-L2, B7-H3, and B7-H4. Finally, B7-H3 and B7-H4 expression was mutually exclusive  and lower naive (p < 0.0001) and CM (p < 0.0001) cells than CD4+ cells  and were rarely polarized as naive or effector cells. CD8+ cells displayed significantly higher late EM frequencies (4+ cells .p < 0.0001), while CD8+ expressed higher PD-1 levels  and iEM CD8+  T cells . By contrast, on CD8+ cells, the CM and iEM subpopulations expressed the highest PD-1 levels, and late EM cells displayed the lowest levels  and lower CD4+ T cells ; and (p = 0.016) and late EM (p = 0.022) T cells. Similarly, naive CD8+ T cells were poorly represented in B7-positive AMLs ; and  higher pive AMLs .p < 0.0001, iEM: p = 0.009, lateEM: p < 0.0001) and PD-1  levels on the effector CD4+, but not CD8+ cells  and was mostly characterized by the presence of molecules with known or most probably inhibitory role in cancer and AML in particular  , 44. B7.NPM1 mutated AML. ICOS-L was the only co-stimulatory ligand that was identified in 40% of B7 signatures. However, its facilitating role is probably either outbalanced by the co-expression of inhibitory ligands PD-L1 or B7.2 or, according to literature data, is detrimental in itself for successful antileukemic immunity by inducing Treg expansion  (data not shown).In complex karyotype AML-NOS, the B7 phenotype was rather restricted to isolated B7.2 expression or the co-expression of B7.2, PD-L1, and ICOS-L, which was also the most frequent B7 signature. On the other hand, normal karyotype AML-NOS cases expressed more diverse B7 ligands, including PD-L2 and B7-H4, which were statistically correlated, but also B7-H3. Interestingly, B7-H3 and B7-H4 expression was mutually exclusive, and B7-H3 expression was correlated with xpansion , PD-1 exxpansion , 48. Brixpansion , we alsoThe B7-negative patient group was smaller (40%) and encompassed most of the AML cases with recurrent genetic anomalies, most notably with t and t.The expression of B7 ligands on AML blasts has been further mirrored by polarization differences in T cell populations and ICR expression. When the B7+ and the B7– cases were analyzed separately, it turned out that the B7+ patient cases had significantly higher cytotoxic and lower T helper cell percentages, while this ratio was reversed within the B7– group. When analyzing the maturation subsets, we were able to show that most of the CD4+ T cells fall in the CM category, unlike the CD8+ T cells where the effector subsets outnumber the central memory ones. Furthermore, B7+ patients had a significantly lower number of naive CD4+ and CD8+ T cells than the B7- patients . The simOn an overall analysis of our AML patients, setting aside the maturation polarization, the ICOS expression was predominant on CD4+ T cells, while PD-1 was higher on CD8+ cells. When compared to B7 negatives, B7-positive patients expressed higher levels of both ICOS and PD-1 on CD4+ T cells, unlike the CD8+ cells that expressed similar levels of ICOS and PD-1 regardless of the B7 positivity or negativity. Despite the significant predominance of CD8+ T cells in B7+ AML patients, it so seems that B7 checkpoint ligands are rather impacting the immunoediting of CD4+ BM T cells.Analyzing further the ICOS and PD-1 expression on maturative subsets, we have noted a progressive increase in PD-1 expression from the naive toward the CD4+ effector compartment, while ICOS expression was highest on lateEM cells. By contrast, PD-1 expression on CD8+ cells was highest on CM, but not effector cells that displayed higher ICOS levels. Since CD4+ T cells promote the CD8+ T cell antitumor activity and prevent their exhaustion , we can When extending the investigation of ICOS and PD-1 expression on maturative subsets in B7-positive and B7-negative patients, we could stress further that the highest ICOS expression was present on late EM CD4+ T cells, followed by CM and iEM CD4+ T cells, and it always surpassed the ICOS expression on CD8+ T cells. Even though the level of expression was constantly higher on the cells of B7-positive patients, no statistically significant differences vs. negative ones or healthy donors emerged. PD-1 was instead always expressed at higher levels on CD8+ T cells as compared to CD4+ T cells, with the highest level reached by the CM CD8+ T cells, followed by iEM and late EM CD8+ T cells. However, the differences between B7+ and B7– patients and healthy donors were again not significant, even though iEM and late EM CD8+ T cells constantly displayed higher levels of PD1. As ICOS and PD1 binding mediate activating, respectively, inhibitory signals, these results might suggest that, irrespectively of B7 expression on AML blasts, CD8+ T cells are more prone to PD-1-mediated apoptosis, while their CD4+ counterparts are more susceptible to activation, but also to activation-induced cell death. Furthermore, the presence or absence of the B7 molecules on the AML blasts seems to have a minimal impact in influencing the levels of expression of ICOS and PD-1 on the T cell surfaces.NPM1 mutated AML, that displayed features holding indirect evidence of an “inflammatory” microenvironment, including the expression of mainly inhibitory B7 ligands, correlated with higher percentages of CD4 effector cells, less CD4+ and CD8+ naive T cells, as well as higher ICOS and PD-1 expression. NPM1 mutated AML also presented effector differentiation of CD8+ PD-1+ T cells, which is likely a feature of immune exhaustion. The rather inflammatory polarity of the immune microenvironment in NPM1 mutated AML is further supported by research that revealed that NPM1 mutation generates immunogenic peptides  but also in AML harboring the t9,11), an entity with rather low immunogenicity, as shown by previous research , 54. Thu,11p21;q.TP53 mutation and adverse karyotypes. In our similarly sized group of newly diagnosed AML patients, we were able to confirm this association between the expression of B7 ligands, including PD-L1 and the adverse-risk ELN patient subgroup.It is most clear that further research is needed to improve the characterization of the T cell composition and immune checkpoint landscape of the BM microenvironment. Recently, a comprehensive study conducted by Williams et al.  addresseWilliams et al.  did not Our study has obvious limitations, including the deliberate simplification of the leukemia immune biology, which depends on many other factors, such as functional T helper polarization , NK cells, macrophages or myeloid derived suppressor cells, and a relatively small patient group made of exclusively newly diagnosed AML cases however reflecting the heterogeneous AML patient population and confirming several conclusions emerging from the study of Williams et al. .Our data provide a more in-depth insight on the immune biology of AML. The B7 expression and antileukemic immunity in general are continuously regulated by a plethora of factors, including tumor-independent ones . Hence, All in all, our results reinforce the concept that this genetically heterogeneous disease has distinct and versatile patterns of antitumor immune response that depend on factors beyond the intrinsic genetic traits of the tumor cells. This finding is particularly relevant since new AML drugs are being rapidly developed and immune profiles emerge as a powerful biomarker in guiding and personalizing the new immunotherapy approaches.Gene expression profiling of the leukemia immune microenvironment is laborious, time-consuming, and not widely available. Hence, alternative markers for a faster and less expensive evaluation of the AML immune landscape are needed. In this paper, we have demonstrated that B7 checkpoint receptor and ligand profiling by flow cytometry is able to generate relevant data.In our study group, in more than half of the cases, the AML blasts expressed B7 molecule signatures or isolated B7 ligands. Regardless of the B7 combination, a co-inhibitory B7 molecule, such as B7.2, PD-L1, or PD-L2, was always present. B7 molecule expression was more frequent in intermediate and adverse ELN risk AML (78.2%) when compared to favorable risk cases (28.5%). Finally, B7 positivity was correlated with primary refractory AML and reduced overall survival.B7-positive AMLs displayed a predominance of effector BM CD4+ T cells with significantly higher levels of ICOS and PD-1. B7-negative AMLs were characterized by a significant predominance of naive CD4+ and CD8+ T cells and lower CD4+ T cell ICOS and PD-1 levels.As B7 ligands and their counterpart T cell receptors are regarded as indirect indicators of an IFN-driven “inflammatory” microenvironment, we can thus hypothesize that the B7 immune profiling of AML blasts and T cells could serve as a useful biomarker to rapidly discriminate between AMLs with “inflamed” vs. “non-inflamed” microenvironment. Rather than defining an AML subtype in itself, the B7 phenotype more likely offers a momentary perspective of the versatile interaction between leukemia and immune cells that could predict resistance to standard chemotherapy and could guide personalized immunotherapy.We have undoubtedly entered a new therapeutic era in AML, where new and effective drugs are being rapidly designed, but the progress of immunotherapy clinical trials has a tendency to outpace our understanding of leukemia immune biology. The immune profiling of the tumor microenvironment will likely have a major impact on future clinical trial design, drug development, and integration of personalized immunotherapy in current therapeutic strategies.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania. The patients/participants provided their written informed consent to participate in this study.IA, MZ, and PC are responsible for the study design. IA, AD, and CD were involved in the clinical management of the patients. MZ, II, and AS performed the immunophenotypic, molecular, and cytogenetic analyses. MP performed the statistical analysis and contributed to the graphical illustrations. IA and PC wrote the manuscript. All the authors were involved in the revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cannabis sativa L.) is currently one of the most controversial and promising crops. This study compared nine wild hemp (C. sativa spp. spontanea V.) accessions with 13 registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain belonging to C. sativa L. The first three groups had similar main essential oil (EO) constituents, but in different concentrations; the CBD hemp had a different EO profile. The concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: β-caryophyllene 11–22% and 15.4–29.6%; α-humulene 4.4–7.6% and 5.3–11.9%; caryophyllene oxide 8.6–13.7% and 0.2–31.2%; and humulene epoxide 2, 2.3–5.6% and 1.2–9.5%, respectively. The concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil while CBD in the EO of the registered cultivars varied from 7.1 to 25%; CBD in the EO of the breeding lines and in the CBD strain varied from 6.4 to 25% and 7.4 to 8.8%, respectively. The concentrations of δ9-tetrahydrocannabinol (THC) in the EO of the three groups of hemp were significantly different, with the highest concentration being 3.5%. The EO of wild hemp had greater antimicrobial activity compared with the EO of registered cultivars. This is the first report to show that significant amounts of CBD could be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. The amount of CBD in the EO can be greater than that in the EO of the USA strain used for commercial production of CBD. Furthermore, this is among the first reports that show greater antimicrobial activity of the EO of wild hemp vs. the EO of registered cultivars. The results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the EO and possibly CBD-rich EO production.Hemp ( Cannabis sativa L.) is a new-old crop, one of the most controversial and promising crops due to its multiple utilizations, and contains a wide array of biologically active substances synthesized and accumulated in different plant parts [C. sativa spp. spontanea Vavilov, also known as spontaneous), which is native to both Central and Eastern Europe as well as parts of Asia, and is found as a weed in agricultural fields.Hemp  can be extracted using various extraction methods, the simplest and most natural using either steam or hydro-distillation, as is the case with many other EO crops. Hydro-distilled or steam distilled hemp EO are generally preferred by consumers and can be incorporated into a number of certified organic products; the market for organic food and non-food products reached US$55 billion in 2019 in the USA alone . That acHemp terpenes in the EO contribute to the aroma of various cannabis genotypes, and so far, around 140 different terpenes have been reported in this plant ,7,8. Cur2, or solvent extraction) [The hemp EO profile depends on genotype, growth conditions, and extraction method  . Of the raction) . These Eraction) ,10. Monoraction) . Overallraction) . Hydro-dS. aureus, H. pylori, Candida, and Malassezia spp., enzymes, and cancer cell lines [+ and G- bacteria and yeast, and the effect depended on the cultivar and seeding date [Hemp EO  has shown biological activity against several targets of pharmaceutical interests ll lines . The EOsing date .C. sativa spp. spontanea) sampled from agricultural fields in northeastern Serbia with 13 EU registered cultivars, eight breeding lines, and one CBD hemp strain (belonging to C. sativa) with respect to their EO profile and antimicrobial activity.This study addresses a knowledge gap and current industry interest towards hemp EO with different origins and profile. The hypothesis was that wild hemp would have a different EO content, composition, and antimicrobial activity compared with the EOs of registered industrial hemp cultivars, new hemp breeding lines, and a hemp strain (unregistered cultivar) that is currently used for the commercial production of CBD. The objective of this study was to compare nine wild hemp accessions  were randomly selected among the nine locations  to be used as two replications to represent wild hemp in the statistical analyses. As indicated in the Materials and Methods section, one-way ANOVA was completed to determine the significance of differences between the mean constituents obtained from nine hemp cultivars from Northeast Serbia. The constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5α-ol, caryophylla-4(12),8(13)-dien-5β-ol, 14-hydroxy-(Z)-caryophyllene, β-bisabolol, α-bisabolol, CBD, and δ9-tetrahydrocannabinol.Overall, the EO profile of the wild hemp was different from that of one of the registered cultivars and the new breeding lines . The EO The concentration range of α-pinene (bicyclic monoterpene) in the EO was from non-detected (n.d.) in three accessions of wild hemp to 2.5% in wild hemp Slavka; from n.d. to 8.4% in the registered cultivars; and 0.15 to 2.95% in the new breeding lines . HoweverThe concentration range of γ-caryophyllene in the EO was 0.2 to 1.23% in wild, 0.6 to 1.4% in the registered cultivars, and 0.57 to 1.6% in the EO of the breeding lines . γ-CaryoC. sativa EO caryophyllane- and humulane-type sesquiterpenes that include sesquiterpenes (E)-β-caryophyllene, (Z)-β-caryophyllene, caryophyllene oxide, and the ring-opened isomer α-humulene (α-caryophyllene) [The concentration of β-caryophyllene was 15 to 30% in the EO of wild hemp, 22 to 55% in the registered cultivars, and 11 to 22% in the EO of the breeding lines . Overallhyllene) . This EOhyllene) .The concentration of α-(E)-bergamotene (bicyclic sesquiterpene) ranged from 0.75 to 1.9% in the wild hemp, 0.36 to 4.4% in the registered cultivars, and 0.5 to 2.8% in the breeding lines . OverallThe concentration of caryophyllene oxide (bicyclic sesquiterpenoid) was 0.24 to 31% of the total EO in wild hemp, 3.9 to 6.8% in registered cultivars, and 8.9 to 17% in the EO of the breeding lines . The conThe concentration of humulene epoxide 2 (bicyclic sesquiterpenoid) was 1.3 to 3.2% in wild hemp, 0.4 to 2.3% in registered hemp cultivars, and 2.3 to 5.6% in the EO of the breeding lines . OverallSelina-6-en-4-ol (bicyclic sesquiterpenoid) was 0.23 to 1.6% in the EO of wild hemp, n.d. to 1.8% in the EO of the registered cultivars, and 1.1 to 2.8% in the EO of the breeding lines . Its conThe concentration of caryophylla-4(12),8(13)-dien-5β-ol (bicyclic sesquiterpenoid) was 1.1 to 5.4% in the EO of wild hemp; from n.d. to 2.3% in the EO of the registered hemp cultivars; and 2.3 to 6.6% in the EO of the breeding lines . Its conThe concentration of β-bisabolol (monocyclic sesquiterpenoid) was 0.9 to 4.5% in the EO of wild hemp; from n.d. to 1.8% in the EO of the registered cultivars; and 2.9 to 4.0% in the EOs of the breeding lines ; Table 2The concentration of α-bisabolol (monocyclic sesquiterpenoid) was 0.4 to 2.9% in the EO of wild hemp, n.d to 6.9% in the registered cultivars, and 0.5 to 3.5% in the EO of the breeding lines . α-BisabThe major EO constituents of the USA hemp strain that was grown in close vicinity had a different chemical profile, with major constituents of myrcene (9.2 to 12%), β-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), β-(E)-ocimene (5.3 to 5.6%), and α-bisabolol (3.9 to 4.4%). Some previous reports identified a different number of EO constituents in hemp EO  ; 84 EO cC. sativa [Previous research has suggested that metabolic fingerprinting can be used for chemotaxonomic purposes in . sativa . However. sativa ,18 have In this study, the concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil, the CBD in the EO of the registered cultivars was from 7.1 to 25.4%, while the CBD concentration in the oil of the breeding lines was from 6.4 to 25.4% . HoweverOverall, the concentration of THC in the EO was significantly different between the three groups of hemp . The THCOverall, the content of monoterpenes fluctuated from n.d. to 8.0% in the EO of wild hemp, 0.3 to 13% in the EO of the registered cultivars, and 0.2 to 10% of the EO of the breeding lines . MonoterSesquiterpenes were the largest group of chemical constituents. The content of sesquiterpenes was 41 to 79% of the EO of wild hemp, 65 to 89% of the EO of the registered cultivars, and 70 to 89% of the EO of the breeding lines . Sesquit9-THC %), however, they were selected to have <0.3% total THC in dried biomass in order to be compliant with the current rules and regulations that are evolving [Cannabinoids also comprised the second largest group of chemical constituents in the wild hemp. The concentration of cannabinoids was 7 to 56% of the EO of wild hemp, 4.6 to 28% of the EO of the registered cultivars, and 6.4 to 26% of the EO of breeding lines . Cannabievolving .In a study of spontaneous hemp, Nagy et al.  reportedThe pharmacological power of hemp is based on the content of Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) . Other m-) bacteria and gentamicin for Gram-positive (G+) bacteria, and fluconazole for yeast. From G- we used SE, Salmonella enterica subsp. enterica; PA, Pseudomonas aeruginosa; and YE, Yersinia enterocolitica. From G+, we used SA, Staphylococcus subsp. aureus; EF, Enterococcus faecalis; and SP, Streptococcus pneumoniae. From yeast, CA, Candida albicans; CK, Candida krusei; and CT, Candida tropicalis were used. The method has been described previously [We used antibiotics as a positive control: cefoxitin for Gram-negative  activity to the antibiotics gentamycin, cetofoxin, and fluconazole.Staphylococcus subsp. aureus (SA), followed by the EO of wild hemp Saykaj . The EOs of wild hemp Buro and cv. Dioica were the most potent against Streptococcus pneumoniae (SP) (Streptococcus pneumoniae compared with that of other EOs (The antibiotic gentamycin was used as a positive control in the data presented in p Saykaj A. Similaiae (SP) B. The EOther EOs C.Pseudomonas aeruginosa compared with that of the EOs of the other hemps , the bioactivity of EO of wild hemp Perez was not significantly different, while the bioactivity of the EOs of the other hemp EOs was lower than that of Saykaj (Candida krusei (CK), while the bioactivity of the EO of Sequieni was not different from the above  (The antibiotic fluconazole was the positive control for the data presented in f Saykaj A. The EOhe above B. The EOlis (CT) C.S. aureus, H. pylori, Candida and Malassezia spp., enzymes, and cancer cell lines [Previously, hemp EO has shown biological activity against several targets of pharmaceutical interest: ll lines . In anotll lines .Overall, the findings in this study are consistent with the ones in a recent report . This stCannabis sativa L.) seeds were obtained from the Institute for Field and Vegetable Crops in Novi Sad, Serbia. Field experiments were set up at the Alternative Crops and Organic Production Department in Backi Petrovac, Serbia (N502138 E395689) using 13 different cultivars of industrial hemp  was applied as a broadcast treatment at 300 kg/ha before deep plowing. An additional 50 kg/ha of nitrogen was applied in the spring before sowing. Weed control was conducted using burnout with glyphosate prior to seeding. Mechanized weed control was performed twice during the first four weeks of vegetation stage though the use of sweep-type cultivators. Hemp closes the canopy at 5-6 weeks after emergence and hence, suppresses weeds very well after that. The trial of the breeding lines was at the same field, approximately 60 m from the main trial, and was subjected to the same agricultural conditions. Plants in both trials were cultivated the same way including seeding time, seeding depth, fertilization, and weed control.Fresh biomass samples  were obtained on 27 June, 2019 at the beginning of flowering of the male plants. Hemp tissue samples were generated by cutting the top 1.5 feet (46 cm) from the top of female plants . Fresh weight was measured, then the samples were hung in a shady area (tobacco dryer) until constant air-dried weight and then extracted.The EO extraction was conducted via hydro-distillation of hemp air-dried material using 4-L hydro-distillation Clevenger-type units as described previously for another plant material . The samm/z range of 40–400. The injector and detector temperature (FID) was set at 220 °C and 280 °C, respectively.Hemp volatile compounds were analyzed by GC-FID and GC-MS as described previously , with th8 to C40 under the same conditions described above.All constituents present in the EO samples were identified by comparing their linear retention indices (LRI) and MS fragmentation patterns with those from the National Institute of Standards and Technology (NIST′08) and Adams mass spectra library. The estimated LRI were determined using a mixture of a homologous series of aliphatic hydrocarbons from CPseudomonas aeroginosa (CCM 1959), Salmonella enterica subsp. enterica (CCM 3807), Yersinia enterocolitica (CCM 5671); Gram-positive bacteria Enterococcus faecalis (CCM 4224), Staphylococcus aureus subs. aureus (CCM 4223), Streptococcus pneumoniae (CCM 4501); and yeasts Candida albicans (CCM 8186), C. krusei (CCM 8271), and C. tropicalis (CCM 8223) . The bacteria cultures were incubated in Mueller Hinton broth  at 37 °C, but yeast cultures were in Sabouraud broth  at 25 °C overnight.The microorganisms used for antimicrobial activity testing included Gram-negative bacteria 6 CFU/mL bacterial suspension after incubation was spread on the Mueller Hinton Agar . Filter paper discs (6 mm in diameter) were infused with 15 µl of the EO, tested, and placed on the inoculated MHA. MHA was kept at 4 °C for 2 h and then at 37 °C for 24 h. For yeasts, 100 µL of the yeast suspension was spread on Sabouraud agar  and agars were cultivated at 25 °C for 24 h. After the incubation period, the diameter of inhibition zones was measured (mm). Growth inhibition was compared with the standard drugs. The standard drugs cefoxitin for G− bacteria, gentamycin for G+ bacteria and fluconazole for yeasts were used as positive controls. Tests were performed in three separate experiments, and the means were calculated.For the agar disc diffusion method, a 100 µL of 10One-way analysis of variance (ANOVA) with two replications was completed to determine the significance of differences among the mean oil content and constituents obtained from nine cultivars. The constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, caryophyllene oxide, humulene epoxide 2, selina-6-en-4-ol, caryophylla-4(12),8(13)-dien-5α-ol, caryophylla-4(12),8(13)-dien-5β-ol, 14-hydroxy-(Z)-caryophyllene, β-bisabolol, α-bisabolol, CBD, and δ9-tetrahydrocannabinol (Dronabinol). This completely randomized design has two replications.The differences among the mean antimicrobial activities  obtained from the seven registered cultivars  and seven wild accessions  were also compared using one-way ANOVA with three replications.p-value < 0.05) or marginally significant  on most of the constituents, further multiple means comparison was completed using the lsmeans statement of Proc Mixed  at 5% level of significance and letter groupings were generated. The overall mean was calculated for the constituents where cultivar effect was not significant. For the antimicrobial activities, cultivar effect was highly significant  on all nine activities, and considering the large number (14) of cultivars and accessions compared, multiple means comparison was done using Tukey’s multiple range test, which controls the Type II experiment-wise error rate.The analyses were completed using the mixed procedure of SAS . Since tFor each response variable, the validity of model assumptions was verified by examining the residuals as described in Montgomery .Cannabis sativa var. spontanea Vavilov, (a synonym of Cannabis sativa L.), considered native to Central and Eastern Europe and parts of Asia. However, the taxonomy of hemp is still debatable and there is no consensus among taxonomists as to whether it is a single species with several subspecies or multiple species.The results from this study demonstrated that the essential oil (EO) of wild hemp from Serbia is different in its chemical profile and bioactivity from the EO of registered industrial hemp cultivars, the breeding lines, and the hemp strain grown for CBD production in North America. Wild hemp EOs were also somewhat different from the EO profile of wild and spontaneous hemps collected in Austria and Hungary, as reported in the literature. However, although having been named differently, the wild hemp in this study, the wild hemp collected in Austria, and the spontaneous hemp from Hungary might have common origins; the collection sites were approximately in the same region (with a dimeter of approximately 500 km) although collected in three different countries. These wild/spontaneous hemps have been exposed to significant environmental and agricultural (pesticide) pressure over the last few decades. These populations may all belong to The concentration of the four major constituents in the industrial hemp lines and wild hemp varied as follows: β-caryophyllene 11 to 22% and 15.4 to 29.6%; α-humulene (α-caryophyllene) 4.4 to 7.6% and 5.3 to 11.9%; caryophyllene oxide, 8.6 to 13.7% and 0.2 to 31.2%; humulene epoxide 2, 2.3 to 5.6% and 1.2 to 9.5%, respectively. The major EO constituents in the USA hemp strain that was grown in the vicinity of the field trials had different chemical profiles, with the major constituents myrcene (9.2 to 12%), β-caryophyllene (6.5 to 7.5%), limonene (3.8 to 4.2%), β-(E)-ocimene (5.3 to 5.6%), and α-bisabolol (3.9 to 4.4%).Overall, the EO of wild hemp has shown greater antimicrobial activity against Staphylococcus susp. aureus, Enterococcus faecalis, and G−Yersinia enterocolitica, Salmonella enterica subsp. enterica, and yeasts Candida albicans and Candida tropicalis compared with the EO of registered cultivars.This is the first report to show that a significant amount of CBD can be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. One of the wild hemp EOs showed a very high concentration of CBD.The EO of the wild hemp had a significantly higher concentration of THC relative to the one from the registered EU cultivars of industrial hemp breeding lines. In addition, it was interesting to see that wild hemp had a higher concentration of CBD in the EO relative to the EO from a strain grown for commercial production of CBD in the USA Therefore, wild hemp collected in Serbia could be used for the development of varieties (registered cultivars) with specific desirable chemical composition, and may also provide excellent material for the selection and breeding of hemp cultivars with high CBD for the commercial production of CBD and other high-value chemicals.


Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats. In the world, the number of people aged 60 and over has been growing faster than any age group Desa, . However2 per week for 5 weeks improved the cognitive function and EEG rhythms of older adults with memory complaint. Using D-galactose-induced aging mice, Salehpour and collaborators  and all efforts were made to minimize animal suffering in accordance with the proposals of the International Ethical Guideline for Biomedical Research  were used in this study. The colony room was maintained at 21 ± 2°C with a 12 h light/ dark schedule (light: 7 am until 7 pm), and food and water were provided n = 6) and control (n = 5). One week before the treatment protocol, the animals were adapted to manual handling (with no anesthesia). This procedure was adopted to minimize discomfort during treatment. After this, the animals from the laser group were manually immobilized and received treatment with a laser diode of 810 nm wavelength and 100 mW power for 30 s (3 Joules of energy/point) by approximation at each of the 5 irradiation points of application . The daily laser treatment total was 15 Joules of energy, 150 s of irradiation, and fluency of 535.7 Joules/cm2. Also, no difference in scalp temperature measured in the animals was noted with a non-contact thermometer during the treatment protocol, corroborating with the results of Wang and collaborators : 810Operating mode: CWAverage radiant power (W): 0.1Aperture diameter (cm): 0.62): 3.571Irradiance at aperture (W/cmBeam shape: Circular2): 0.028Beam spot size (cm2): 3.571Irradiance at target (W/cmExposure duration/point (s): 302) per session: 535.7Radiant exposure  per head: 15g for 10 min at 4°C and supernatants were transferred to a new tube.Twenty-four hours after the final laser/placebo session (59th day of the experiment), the rats were euthanized by decapitation and their cerebral cortex  and hippocampus (Ammon’s horn and dentate gyrus) were immediately collected and frozen. The tissue samples were homogenized in ice-cold RIPA lysis buffer  with freshly added protease  and phosphatase  inhibitors. Homogenates were centrifuged at 10,000× ® MAP kits magnetic bead panel assay  following the manufacturer’s specifications. This multiplex immunoassay allows the simultaneous quantification of the expression and activation of cortical and hippocampal proteins: Akt, p70S6K, STAT3, STAT5, ERK, JNK, and p38. The plates were read on a LuminexTM MagpixTM instrument and results were analyzed with the Milliplex Analyst 5.1 Software using a Logistic 5P Weighted regression formula to calculate sample concentrations from the standard curves.The expression and activation  of intracellular proteins in the brain samples were quantified using the MilliplexP-value was lower than 0.05. All plots were acquired using the Graph Pad Prism (6.0). Data are presented as individual points to show dispersion and as mean and standard error of the mean (±SEM).Statistical procedures were conducted using the Mann-Whitney U-Test for independent group comparisons of nonparametric data. The Z-score was used to remove outlier values (−/+2 SD). In the cortex, we removed one outlier of p-ERK/ ERK from the control group and one outlier of p-p38/p38 from the laser group. In the hippocampus, we removed one outlier of STAT3 and STAT5 from the control group and one outlier of p-ERK/ERK from the laser group. All analyses were performed using the Statistical Package for the Social Science . A statistical difference was considered significant when the two-tailed The expression and activation of intracellular signaling proteins  were investigated in the cerebral cortex and hippocampus of rats from the laser and control groups. The cortical and hippocampal expression and activation are presented in , 2. The U = 8.000; p = 0.201), p70S6K , STAT3 , STAT5 , ERK , JNK  and p38 . With regard to the activation of signaling proteins , no significant difference was observed between the groups studied in the cortical activation of Akt , p70S6K , STAT5  and p38 . However, PBM significantly increased the cortical activation of STAT3 , ERK  and JNK , STAT5 , ERK , JNK  and p38 . However, PBM significantly increased the hippocampal expression of p70S6K  and STAT3 , STAT3 U = 12.000; p = 0.917), STAT5  ERK , JNK  and p38 . However, PBM significantly increased the hippocampal activation of Akt , while our protocol consisted of 58 consecutive days of laser treatment with 810 nm wavelength and fluence of 535.7 J/cm2. Therefore, our laser treatment was 10 times greater. PBM is well known to show hormetic dose-responses, with opposite effects at lower and higher doses . These results suggest a neuroprotective effect of PBM since these proteins are linked to cell survival, apoptosis, and protein synthesis .Conceived and designed the experiments: FC, FM, and SG. Performed the experiments: FC and FM. Analyzed the data: FC and SG. Contributed reagents, materials, and analysis tools: FC and SG. Wrote the manuscript: FC, RL-M, FG-L, and SG. Approved the final version of the manuscript: FC, FM, RL-M, FG-L, and SG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Little is known about how cancer cells adapt their mechanical properties in complex 3D microenvironments. Here we generated different types of tumor spheroids within compliant or stiff hydrogels. We then quantitatively mapped the mechanical properties of these spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiffer hydrogels showed elevated Brillouin shifts, hence spheroids became “stiffer” compared to the ones cultured within compliant gels. The spheroid’s mechanical properties were modulated by various microenvironment properties including matrix stiffness and degradability and the resultant compressive stress but also depending on whether single cells or cell aggregates were analyzed. Moreover, spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels became stiffer and at the same time, less invasive compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and their microenvironment, which is relevant to better understand cancer progression.Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts  with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression. Tumor cells reside within a complex microenvironment that regulates essential cellular functions and also plays an important role in tumor progression and metastasis ,2. The uSince increased ECM stiffness has been correlated with tumor aggressiveness ,12,13, iMulticellular 3D cell culture models such as tumor spheroids recapitulate relevant features of epithelial tumors, including more realistic cell-cell contacts, proliferation rates, diffusion gradients and drug responses ,26,27. WFor this reason, we set out here to investigate the mechanical properties of tumor spheroids in situ using Brillouin microscopy (BM) . It is aWe report here that different types of tumor spheroids growing in stiff degradable hydrogels are characterized by a larger Brillouin frequency shift, hence increased elastic moduli, as compared to spheroids in compliant degradable hydrogels. This indicates that the spheroids adapted their mechanical properties to their microenvironment. Systematically comparing different culture systems where differences in compressive stress occur, e.g., after blocking hydrogel degradation, probing single cells or free spheroids, revealed a positive relationship of increasing Brillouin frequency shifts and compressive stress. Spheroids formed from invasive breast, prostate and pancreatic cancer cells formed in stiffer degradable hydrogels, showed higher Brillouin shifts and were less invasive compared to those in compliant degradable hydrogels. Thus, we find that the spheroids’ mechanical phenotype and cell invasiveness are modulated by matrix stiffness, matrix degradability, multicellularity and concomitant changes in compressive stress levels. Together, our results bring new insights into the mechanical interactions between tumor cells and their microenvironment, which is relevant to better understand cancer progression.® was used. All cell culture reagents were from Thermofisher unless otherwise stated.MCF-7, MDA-MB-231 and PC3 cells were obtained from the “Deutsche Stammsammlung für Mikroorganismen und Zellkulturen” (DSMZ). PANC1 cells were kindly provided. MCF-7 cells were maintained in RPMI 1640  supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, MEM non-essential amino acids, 10 µg/mL human insulin  and penicillin (100 U/mL) -streptomycin (100 µg/mL). MDA-MB-231, PANC1 and PC-3 cells were cultured in DMEM/F-12, DMEM-GlutaMAX and RPMI 1640 respectively, supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL) -streptomycin (100 µg/mL). Before starting 3D cultures, cells were grown in standard T25 culture flasks and sub-passaged 2–3 per week. For cell detachment, TrypLE®, resuspended in cell culture medium, centrifuged down, and resuspended in PBS. Then they were mixed with the freshly prepared heparin-maleimide (MW~15000) solution and RGD peptide , at a final density of 5.6 × 105 cells/mL and a final heparin-maleimide concentration of 1.5 mM. For a molar ratio, PEG/heparin-maleimide of gamma = 1.5, PEG precursors (MW~16000) were reconstituted with PBS at a concentration of 2.25 mM and placed into an ultrasonic bath (Merck) for 20 s (medium intensity). Lower stiffness gels were prepared by diluting the stock solution of PEG precursors accordingly (1:2 for gamma = 0.75). To prepare PEG-heparin hydrogel droplets, heparin-cell suspension was mixed in a chilled microtube with the same amount of PEG solution using a low binding pipette tip. Then, a 25 µL drop of the PEG-heparin-cell mix was pipetted onto hydrophobic, Sigmacote® (Sigma Aldrich)-treated glass slides (VWR). Gel polymerization started immediately, and stable hydrogels were obtained within 1–2 min. Hydrogels were gently detached from the glass surface after 4 min using a razor blade and transferred into a 24 multi-well plate supplemented with 1 mL cell culture medium. Cell culture medium was exchanged every other day.PEG and heparin-maleimide precursors were synthesized as previously described . Cells wAfter their preparation, hydrogels were immersed in PBS and stored at 4 °C until mechanical characterization on the following day. After equilibrating them at RT for 1 h, gels were mounted using CellTak  onto glass object slides . A Nanowizard I or IV  was used to probe the gel stiffness. Cantilevers  that had been modified with polystyrene beads of 10 μm diameter  using epoxy glue , were calibrated using the thermal noise method implemented in the AFM software . Hydrogels were probed at RT in PBS using a speed of 5 μm/sec and a relative force setpoint ranging from 2.5 to 4.0 nN in order to obtain comparable indentation depths (0.5–1 µm). Force distance curves were processed using the JPK data processing software . Indentation parts of the force curves (approximately 1.5 μm indentation depth) were fitted using the Hertz/Sneddon model for a spherical indenter, assuming a Poisson ratio of 0.5 ,43.formaldehyde/PBS for 45 min, followed by a 30 min permeabilization step in 0.2% Triton-X100. Spheroids were stained for 2–4 h with 5 µg/mL DAPI and 0.2 µg/mL Phalloidin-TRITC in 2% BSA/PBS. Then hydrogels were immersed at least for 1 h in PBS. Spheroids were imaged with a LSM700 confocal microscope using a 40× objective . Quantitative analysis of cell morphology was done using FIJI. Briefly, a threshold was applied to the fluorescent images , which were then transformed into binary pictures, and shape factor (defined as 4 × π × area/(perimeter)2) and area were calculated.On day 14, cultures were fixed with 4% Brillouin maps were acquired using a custom-built confocal Brillouin microscope employing a two-stage VIPA spectrometer as previously described in ,45. The p < 0.05). As indicated in the figure legends, for pairwise comparisons a Mann–Whitney (non-parametric) test was used since most datasets were not normally distributed. In the case of more than two groups, a Kruskal–Wallis (non-parametric) with multiple comparisons (Tukey) was chosen. To test for normality, a D’Agostino–Pearson omnibus normality test was performed.GraphPad Prism was used to plot data and to perform statistical tests. A two-tailed significance level of 5% was considered statistically significant  spheroids by embedding single cells into PEG-heparin hydrogels and letting them grow to multicellular aggregates A. HydrogAfter seeing a difference in spheroid growth and compaction in response to hydrogel stiffness, we set out to investigate how matrix stiffness would affect the mechanical phenotype of the spheroids in situ. We employed BM A to quanIn order to investigate whether the seen changes in Brillouin frequency shift were a response of compressive stress acting on the tumor spheroids during growth, we next set out to manipulate the levels of compressive stress on growing tumor spheroids. Firstly, we tested how microenvironment degradability would affect the spheroid’s mechanical properties since our bead pressure sensors had indicated that matrix degradability had a large effect on the growth-induced compressive stress H. Therefp = 0.001), with a trend of increasing Brillouin frequency shifts with hydrogel stiffness. Moreover, unconfined spheroids were generated by MCF-7 cell aggregation in low-adhesive U-well plates (p = 0.73 compared to non-degradable compliant). BM on unconfined spheroids , PANC1 (pancreatic cancer cell line) and PC3 (prostate cancer cell line) in compliant and stiff non-degradable gels A–C, all We then set out to explore how these invasive cancer cell lines behaved in a microenvironment that would permit cell invasion. Therefore, we seeded MDA-MB-231, PANC1 and PC3 cells in compliant and stiff degradable hydrogels to generate spheroids. In the compliant degradable hydrogels, MDA-MB-231 cells formed no spheroids as they dispersed through the gel A, which In the presented study, we have investigated the influence of microenvironment stiffness on tumor spheroid growth, morphology, invasion and mechanical properties using a platform combining engineered tumor microenvironments and BM, an emerging tool in the biomechanics field. To investigate spheroid mechanics in 3D microenvironments of defined stiffness, we have chosen in our study a biohybrid PEG-heparin hydrogel system. Our 3D engineered model incorporates different aspects of mechanical cell interactions, such as growth-induced compressive stress, cell-cell, as well as cell-matrix interactions (by including RGD peptides). Importantly the hydrogel’s mechanical properties can be tuned independently of ligand density. Young’s moduli of the predominantly elastic hydrogels, as characterized by AFM, ranged between 1 and 20 kPa and were therefore within the range of elastic moduli reported for breast tumor specimens . When weWhile these results were obtained on hydrogels, also various experiments have been conducted in the past to reveal biological properties that influence the Brillouin frequency shift measured for cells. For instance, drugs that interfere with F-actin polymerization were combined with BM, which resulted in decreased Brillouin frequency shifts in different cell types ,51. AddiWhen we compared the spheroids’ mechanical properties within the BM maps, we found increased Brillouin frequency shifts for spheroids in stiff degradable gels. Plotting Brillouin frequency shift of spheroids versus Brillouin frequency shift of hydrogels, a direct relation was seen , which sUnder invasive conditions, i.e., invasive cell lines in compliant degradable gels, we have found lower overall Brillouin frequency shifts, which might also indicate decreased cell stiffness of invasive cells or result from a looser structure. However, also for PC-3 cells, where a similar cell density was seen in compliant and stiff degradable hydrogels, a lower Brillouin frequency shift was detected for invasive spheroids in compliant hydrogels. Since we suspected that the Brillouin frequency shift for the invading single cells might have been biased by averaging with the surrounding hydrogel within the scattering volume, we decided to look at the maximum values within each map. In compliant degradable gels, we observed lower Brillouin frequency shifts for invading cells compared to no non-invading cells in stiff hydrogels, which fits the picture of invasive cells having decreased stiffness values . In contThe finding that so little invasion occurs in stiff degradable hydrogels appears at first sight contradictory to previous work showing greater invasion in stiffer microenvironments . HoweverAs mentioned before, an important aspect of stiff tumor microenvironments is compressive stress . This coTaken together, our study brings new insights into the mechanical interplay between tumor spheroids and their complex 3D microenvironment. We have shown that the level of compressive stress varies in our system with matrix stiffness and degradability and modulates growth, morphology, invasion and mechanics of tumor spheroids. Increased elastic moduli of spheroids were associated with lower invasion into the matrix, which suggests that the cancer cells’ adaption of their mechanical properties in response to their microenvironments might also play a functional role in cancer progression. Since all these aspects can be modified with our experimental setup, we have a powerful model at hand to next investigate molecular mechanisms regulating the seen cellular responses. Ultimately, more insights into these mechanisms may provide a basis for identifying new targets that could potentially be used to interfere with tumor progression.


Bioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation.Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions.As inoculant we focused on the monoaromatic compound-degrading bacterium P. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies.The online version contains supplementary material available at 10.1186/s40793-021-00378-x. The recovery and restoration of soils polluted by organic compounds may be enhanced by introducing specific biodegrader bacteria –4. This Sphingomonas wittichii RW1 express differently during transition and growth in (polluted) sand as compared to liquid growth with the same carbon substrate [S. wittichii with those of Pseudomonas veronii strain 1YdBTEX2, isolated from sites contaminated with aromatic compounds [S. wittichii RW1, inoculation into sandy soil provoked a major reorganization in global gene expression of P. veronii 1YdBTEX2, implicating one-third of all genes, but with very few pathways and biological processes in common between the two [Previously, we suggested that more attention should be given to the combination of factors representing the actual expected conditions at polluted sites , and theubstrate . A complubstrate . This suubstrate . In ordeompounds , during ompounds . Similar the two .P. veronii 1YdBTEX2 gene expression changes during adaptation to a soil environment we focus here on its growth and establishment. The questions we aimed to address here were whether P. veronii would be capable to grow and survive in natural non-sterile soils, and whether this would be dependent on the presence of contaminated material or specific added carbon substrate (toluene). We further aimed to understand whether P. veronii would react differently in response to soil type and whether its cellular reactions could be revealed from global gene expression changes. In our experimental design, we inoculated P. veronii and measured population growth in three different natural non-sterile soils, in comparison to regular liquid culture and inert silica matrix, in the presence or absence of externally added toluene or in historically contaminated material with (polycyclic) aromatic hydrocarbons. Community RNA was extracted in exponential phase of P. veronii growth and in stationary phase, which was reverse-transcribed, ribosomal RNA depleted, and sequenced (RNA-seq). Adaptation and cellular reactions of P. veronii under different conditions were interpreted from a combination of global tools, including gene ontology (GO) [P. veronii 1YdBTEX2 (iPsvr) [To complement previous studies on ogy (GO)  and clusogy (GO) , a previ (iPsvr) , as wellP. veronii is a toluene, benzene and m- and p-xylene degrading bacterium originating from contaminated soil [P. veronii colony-forming units (CFUs) in soil, we used a derivative with a mini-Tn5 insertion constitutively expressing the green fluorescent protein (GFP) from the Pcirc promoter of the ICEclc element [P. veronii and the tagged strain , and was grown for 48 h at 30 °C. P. veronii colonies were restreaked on 21C-type minimal medium (MM) [v/v) in MM with 10 mM succinate and again incubated as before. For soil growth experiments, cells were recovered from exponentially growing cultures on succinate (at culture turbidity OD600 = 0.4) by centrifugation (4000×g for 5 min at room temperature), resuspended in MM without further carbon source and then transferred to the soil microcosms (see below). Growth of P. veronii was determined by CFU counting of appropriate dilutions on MM plates with toluene (gas phase). P. veronii colonies were differentiated from any background soil bacterial colonies growing on MM plates with toluene by their green fluorescence, and counted under a digital dual band microscope (Dino-Lite model AM4115T-GRFBY) using the 485 nm blue LED excitation and 510 nm emission filter.ted soil . To more element . We founium (MM)  agar plaP. veronii. These consisted of (i) a sandy soil sampled in a lake Geneva beach near in St. Sulpice – named Sand, (ii) a silty soil sampled from the bank of the local stream ‘Sorge’ on the university campus – named Silt, and (iii) a clay soil sampled in a forest area on campus near to Lake Geneva – named Clay. Quantities of ~ 5 kg were spread on aluminium foil and air-dried on the laboratory bench. Sand and Silt were dried for 7 days (further water losses were not evident with longer drying periods), whereas Clay was dried for 15 days. At the moment of microcosm inoculations, the Sand, Silt, and Clay had gravimetric water content (GWC) of 0.14 ± 0.01%, 0.20 ± 0.01%, and 2.22 ± 0.06%, respectively. pH-H2O of the materials was 7.14 ± 0.02 (Sand), 8.57 ± 0.2 (Silt) and 7.78 ± 0.02 (Clay). Total organic matter content amounted to 0.028 (Sand), 0.13% (Silt) and 4.0% (Clay). Total cell counts on washed material and stained by SYBR Green I were quantified according to Weinbauer et al. [Three natural soils were used for microcosm growth studies with r et al. . The eff. According to previous characterization [10 – C40; 9500 mg kg− 1) and polycyclic aromatic hydrocarbons (2300 mg kg− 1). In addition, minor concentrations of benzene (3.3 mg kg− 1) and methylated monoaromatic compounds were found (22 mg kg− 1). The original material consisted mainly of gravel (1–5 cm) covered with a layer of tar-containing mud. In order to obtain more easily handleable material, it was mixed 65/35 (w/w) in small portions with air-dried Silt and sieved through 3–mm diameter to remove the gravel. In the main text we refer to this mixed and sieved material as Jonction. At the moment of inoculation, Jonction had a GWC of 6.55 ± 0.15%.The polluted material (Jonction) originated from a former gasification work in Geneva and was obtained through collaboration with Biotech S.A., Geneva (CH)rization , the matP. veronii miniTn5::gfp population growth in soil was assessed in microcosms artificially contaminated with toluene. Four replicate microcosms were prepared, each consisting of 95 g of dried Sand, Silt or Clay inside 500–ml glass Schott bottles and closed with Teflon-lined screw caps. As control for porosity effects, we used autoclaved quartz at 5% GWC ; hereafter referred to as artificial porous matrix or APM.The kinetics of P. veronii to obtain 2.5 × 104 CFU g− 1 material at the start. Microcosm flasks were mixed on a horizontal roller mixer  at 80 rpm for 30 min, with manual shaking every 5 min to detach the soil from the walls. After mixing, the tip containing the toluene was placed back inside and the bottles were incubated upright at 24–26 °C in the dark for 60–120 h with regular sampling (see below).Toluene was dosed through the vapor phase from a sealed 1–ml micropipette tip, placed inside the glass bottle and containing 0.2 ml of pure toluene. Tubes with toluene were removed before inoculation with 5 ml suspension of washed preculture of P. veronii miniTn5::gfp in Jonction was followed in (triplicate) microcosms of 10 g in 50 ml polypropylene screw-cap tubes  containing either 100% Jonction (see above), 100% Silt, or mixtures of 75% (g g− 1), 50, and 25% Jonction with Silt. Microcosms were again inoculated with washed P. veronii preculture (0.5 ml) to achieve 2.5 × 104 CFU g− 1 at the start. Microcosms were homogenized and incubated as above, either without any further addition of carbon, or amended with a sealed 1 ml micropipette tip containing 20 μl pure toluene (as described above).Growth of P. veronii on toluene in liquid suspended culture was measured from four replicate 100–mL screw-cap conical flasks containing 15 ml MM and starting cell concentrations of ~ 1 × 107 CFU ml− 1. As we found in preliminary experiments that P. veronii growth on toluene in liquid culture was less consistent with gas-phase dosing, we deployed an inert oil-toluene mixture instead. For this, we mixed 1:25 (v/v) of toluene:tetradecane  and added 0.5 ml per 15 ml liquid culture. Flasks were incubated at 30 °C and at 180 rpm on an orbital shaker.Growth of P. veronii CFU as described above. Identities of P. veronii miniTn5::gfp colonies were verified by their GFP fluorescence.Soil and liquid microcosms were sampled directly after inoculation (1 h) and then four times per day at approximately 6 h intervals . Samples of 5 g (or 2.5 g for Jonction) were retrieved from the microcosms with a spatula and transferred to clean 50 ml polypropylene tubes. 10 ml of sterile saline solution (0.9% NaCl) was added to each tube and cells were extracted by vortexing for 1 min. Larger soil particles were allowed to sediment for a few seconds, after which the supernatant was transferred to a fresh 50 ml tube and serially diluted in sterile saline. For liquid cultures, a 1–mL aliquot was taken directly from the flask and serially diluted. Serial dilutions were drop-plated (10 × 5 μl drops of each dilution) on MM agar, which was incubated with toluene vapor to quantify the number of max) of P. veronii were calculated from the slope of the mean log10 CFU g− 1 over time across quadruplicate assays . We further refer to the maximum population size (max pop size) as the highest mean CFU g− 1 or ml− 1 observed during the entire experiment for each condition, averaged from four (soils) or three (Jonction) replicates at the same sampling time point. Growth rates were compared among treatments by ANOVA, followed by post-hoc Tukey testing.Growth rates , during estimated exponential (EXPO) or in stationary (STAT) phase.For genome-wide expression analysis we used 7 ml− 1P. veronii cells into soil microcosms with 95 g material  in 500 ml capped-glass Schott bottles, with toluene dosage through the gas phase. Cells were prepared from exponentially growing liquid cultures as described above, but resuspended in MM with 0.5 mM succinate to avoid starvation [For the transition phase we inoculated 5 ml (2.5 ml for Jonction) of a suspension of ~ 10arvation . We usedP. veronii during exponential growth and stationary phase was sampled from similar inoculated microcosms , but in this case with a low starting cell density of ~ 104P. veronii CFU g− 1 or ml− 1 to achieve sufficient growth in the soils. Cells were precultured as before, but washed and resuspended in MM without succinate. Microcosms were dosed with toluene as before (four replicates each), and sampled (10 g) at approximate exponential and stationary phase for P. veronii based on initial growth experiments as in Fig. P. veronii was inoculated into 100 ml screw-cap conical flasks containing 25 ml of MM amended with 0.5 ml of a 1:19 mixture of toluene:tetradecane to obtain a starting OD600 of 0.16 (four replicates). Flasks were incubated at 30 °C and 180 rpm on an orbital shaker. Cells were sampled after 4 h  and 24 h , and harvested by centrifugation at 3500×g, for 6 min at 30 °C. Pellets were snap-frozen in liquid nitrogen and stored at − 80 °C until RNA extraction.In order to improve yields for RNA extraction and limit humic acid interference, we first washed cells from solid samples of 10 g by adding 20 ml of 4 °C-cold sterile saline solution (0.9% NaCl) to the tube (50 ml volume) and vortexing for 2 min. Suspensions were allowed to sediment for 1 min, after which the cleared liquid was transferred into a clean 50 ml tube, which was centrifuged for 4 min at 4000×g at 4 °C to collect the cells. After centrifugation, the supernatant was immediately discarded by inversion, the excess of liquid from the walls was quickly removed with a clean paper towel, and the cell pellet was snap-frozen in liquid nitrogen and stored at − 80 °C until RNA isolation. Estimated cell recoveries from the washing procedure are reported in Supplementary Figure RNA was extracted from frozen cell samples using the RNA PowerSoil Total RNA Isolation Kit (MoBio Laboratories). Cells in thawed samples were disrupted in a bead-beating protocol as recommended by the manufacturer (MoBio Laboratories). RNA was purified following MoBio procedures and the final RNA pellet was resuspended in a volume of 20 μl of RNase-free water. Contaminating DNA was removed by two consecutive treatments with TURBO DNase (Ambion), followed by purification using an RNeasy MinElute Cleanup kit .P. veronii genome. RNA samples were then depleted from ribosomal RNAs by using the Ribo-Zero rRNA Removal Kit Bacteria protocol . Subsequently, the RNA was reverse-transcribed, indexed and amplified by PCR using the ScriptSeq™ v2 Bacteria kit and ScriptSeq™ Index PCR primers set 1 (Epicentre). The resulting directional RNAseq libraries were sequenced using single 100-nt read chemistry on an Illumina HiSeq 2500 platform  at the Lausanne Genomic Technologies Facility.RNA quantity (reported in Supplementary Table P. veronii genome sequence as reference (European Nucleotide Archive under bioproject number PRJEB11417). A summary of the total number of mapped reads per condition is listed in Supplementary Table P. veronii transition response (1 h) in liquid suspension and in Sand were taken from previous work [Raw reads were quality-filtered, mapped, sorted and indexed with Bowtie2  and Samtous work .p-value of < 0.01 and a log2 fold-change > 2, and were subsequently interpreted by using Gene Ontology (GO) analysis [P. veronii genes were inferred using the program BLAST2GO [Genes were called significantly differentially expressed between two conditions at a false-discovery rate (FDR) of < 0.05, analysis . GO termBLAST2GO . The samBLAST2GO  with nodP. veronii 1YdBTEX2 metabolic capacities was extracted from its reconstructed genome-scale metabolic model (iPsvr), which accounts for 1234 metabolic genes [2 normalized expression attributed to that reaction. Those reactions whose mean expression differed by two or more standard deviations in EXPO phase among cells in liquid, Silt or Jonction, were highlighted in different color. Maps were exported to Adobe Illustrator (vs 2020). The mean normalized expression of all genes attributed to KEGG reactions under all conditions was further visualized as a heatmap with rows  clustered in Euclidian distance .A survey of ic genes  and all ic genes . Identific genes  with linThe raw unmapped RNA-seq reads related to this study have been deposited in the NCBI Short Read Archive under Bioproject accession number PRJNA682712.P. veronii in different soils, we inoculated microcosms dosed with toluene in comparison to liquid suspended medium or to artificial porous medium  and two natural soils (Sand and Silt) were slightly higher and statistically different from those measured in liquid or in APM . Population size variations in Clay suggested alternating growth and decline, perhaps as a result of cycles of predation (given that the soils were not sterilized). Predation may have been more pronounced in the Clay microcosms because of their slightly higher starting water content (Methods), in which protozoan may have remained alive for longer than in air-dried Sand or Silt [In contrast to the other microcosms, the  or Silt –38. Grow1.28 × 10 CFU g− 1P. veronii could grow in field-collected polluted soil. Since contaminated material from Jonction contains monoaromatic compounds (see above) [P. veronii might be able to grow in absence of externally added toluene. Contrary to our expectations, however, P. veronii miniTn5::gfp developed poorer in microcosms with a higher degree of Jonction material but without added toluene  within 24 h, in 100% Jonction it only reached 2.7 × 105 CFU g− 1. Mixing Jonction material with Silt overproportionally reduced the final attained P. veronii population size  Silt, but that the strain is inhibited by components or factors originating from Jonction. The poorer growth in microcosms mixed with Jonction material further suggested that P. veronii found little available aromatic substrates , in order to determine to what extent e above) , we hypoene Fig. . WhereasP. veronii grew very rapidly in all Jonction microcosms to which external toluene was supplied through the gas phase , exponential growth and stationary phase , both in presence of toluene  and metabolism  of putrescine , genes from the nar operon for nitrate respiration (narGJKLY), a gene encoding oxygen-independent coproporphyrinogen-III oxidase (hemN), and genes coding for the cbb3 cytochrome C oxidase isoforms, ccoN1, ccoO and ccoG2 . These effects may thus be solely a response to change in porosity from the growth matrix itself.P. veronii in different soil types  caused a further common core of 68 genes (60 higher and 8 lower) to change expression  involved in malonate transport and metabolism, and of bkdA1A2B, involved in the metabolism of branched-chain amino acids, were higher expressed  and the narGIJKY gene cluster for nitrate respiration were also significantly induced in P. veronii in soils after 1 h  operon, which is responsible for molybdopterin biosynthesis  were also higher expressed in cells in Jonction, suggesting further induction of anoxic respiration pathways.Among the genes induced specifically in Jonction and not in clean soils  and for the phosphate starvation-inducible protein PsiF. The GO enrichment analysis of transcriptomic response in Jonction was coherent with these observations , elongation factors (EF), fusA (EF-G), efp (EF-P), tsf (EF-Ts), and tuf (EF-Tu); (ii) DNA replication; (iii) ATP synthesis; (iv) sulfate transport; (v) TCA cycle, and (vi) amino acid biosynthesis.As expected, both GO analysis and individual gene annotations confirmed most of the commonly higher expressed genes in exponentially growing cells to be related to growth and energy production Table  and 23. In contrast, signatures of commonly lower expressed genes were globally less clear. Around 50% of those encoded hypothetical proteins (Table meta-cleavage metabolism (dmp gene cluster) and urea transport  showed P. veronii during stationary phase conditions and different growth environments. In comparison to 1 h, cells sampled in stationary phase conditions , glycogen (glg), and polyhydroxyalkanoates (phaG), the latter being induced up to 55-fold in APM and 200-fold in Silt , putrescine (spuD), spermidine (spuE), nitrate (nasA), and sulfate (PVE_r1g3919). Genes associated with resistance mechanisms were higher expressed, for example, organic hydroperoxide resistance protein (ohr), and mercury resistance (merACTP). Interestingly, several signaling genes such as cheA, cheB2, cheW, and cheY, PVE_r1g893, and PVE_r1g2555 were also higher expressed in APM and Silt compared to LQ  in controlled conditions of growth under sublethal solute and matric stress that mimicked water stress in soils, in order to identify common strategies [P. veronii transcriptomic response observed here in APM and, previously, under imposed matric and solute stresses [P. veronii with toluene in APM was worse than in liquid or natural soils, suggesting that porous conditions caused differences in the regulation of toluene metabolism in P. veronii.Previous studies had indicated the massive difference in gene expression of criptome . Furtherrategies . By inclrategies , 49, 50.rategies . On the stresses , was difP. veronii, and to understand if there are soil factors that would determine its adaptation and survival. The soils we worked with here have different microbiota background levels, and some showed growth on toluene, which may have invoked substrate competition on the introduced P. veronii cells. Unfortunately, we do not understand the nature of signs for competition in the P. veronii transcriptome. Soils further varied in pH, organic carbon content, structures and textures, leading to expected differences in e.g., substrate and water availability, matric stress, oxygen fluxes [P. veronii and induction of cytochrome c oxidases cbb3, which have been observed as low oxygen environmental responses of P. aeruginosa [Pseudomonas stutzeri A15, and Pseudomonas fluorescens YT01 [P. veronii to adapt to soil microniches with low oxygen concentrations, as previous studies in controlled porous matrices have shown [P. veronii to adapt to living in soil.One of the aims of this work was to study the effect of soil type on physiological responses and growth of n fluxes , or protn fluxes . Indeed,ruginosa , Pseudomens YT01 , 54. Nitve shown . PossiblP. veronii cells to induce an osmoprotective response (putrescine and potassium uptake), to induce genes for exopolysaccharide production, and to regulate genes for copper homeostasis, indicating the importance of these processes for the initial soil adaptation. Of particular interest is the strong upregulation of systems involved in putrescine uptake and metabolism. The roles of putrescine (and other polyamines) are manifold, having been described both as signals for and during carbon or nitrogen starvation, regulation and oxidative stress defense [P. veronii, whose reaction is in line with the three classical physiological responses to osmotic stress: polyamine transport and accumulation of glutamate and potassium [Regardless of the soil type and even in contaminated material such as Jonction, transition caused  defense , and rec defense . The acc defense . Our datotassium . Particuotassium , 58.P. veronii population developed in all non-sterile environments, more extensively when toluene was added as specific growth substrate, but also in its absence. This is in contrast to the much cited incapacity of inoculants to grow and be metabolically active in natural non-sterile materials, which may lead to bioaugmentation failures [P. veronii would not be able to adapt and proliferate, but unfortunately, we could not really exploit this at transcriptomic level. Growth in Jonction material without added toluene was clearly not favorable for P. veronii, but the transcriptomic response from 1 h exposure in Jonction with toluene (in which it could grow) did not particularly show signs of poor adaptation. Except for increased induction of stress defense systems and changes in expression of genes for respiratory activity, there were no particular signs of physiological breakdown in cells in Jonction. The only other environment, which did not lead to strong population development of P. veronii on toluene was Clay. We think that the most likely causes for population loss here were predation and, possibly, competition of native soil microbiota for toluene. Unfortunately, we did not manage to isolate sufficient RNA for transcriptome analysis from P. veronii during exponential growth in Clay. However, during the 1-h transition in Clay we observed a decrease of expression of genes for flagellar assembly and motility, which was not present in Silt or Sand. Flagella allow bacteria to explore their environment, to search for nutrients and to escape from predators or adverse conditions, but activation of flagellar genes have also been implicated in the solvent stress tolerance response, where the flagellar export apparatus is used to export other proteins unrelated to flagellar assembly [S. wittichii RW1 in sand [S. wittichii RW1, Artrobacter chlorophenolicus A6 and P. veronii reduce expression of flagella synthesis under solute and matric stress [P. putida KT2440 exposed to water stress on ceramic plates did not show significant difference in flagellar gene expression [P. veronii during growth in Silt with toluene upregulated flagellar gene synthesis  is necessarily a ‘narrative’ of understanding how cells adapt and grow, concluded from the conglomerate of global  analysis as well as that of individual gene annotations. However, specific cellular reactions are clearly different between typical liquid cultures and in soil. Therefore, it seems crucial to us to study strain behaviour under the conditions of the expected complex environments , and not in standard liquid culture. In the future, this knowledge may help to better predict the success of inoculants.Additional file 1: Supplementary Figure 1. Comparison of growth rates on toluene of P. veronii wild-type and the P. veronii gfp-tagged variant. Supplementary Figure 2. Background soil microbiota counts. Supplementary Figure 3. Cell washing recoveries of wild-type P. veronii cells inoculated to soils. Supplementary Figure 4. Background growth of P. veronii miniTn5::gfp on soil organic carbon or soil microbiota on toluene.Additional file 2: Table S1. Summary of total mapped reads of inoculated microcosms exposed to toluene. Table S2. Subset of genes whose expression levels responded to inoculation into glass beads (APM) versus liquid (FDR < 0.05). Table S3. Subset differentially expressed genes of P. veronii 1YdBTEX2 in all soils vs controls (Liquid&APM). Table S4. Relevant KEGG reactions with outlier expression in exponential phase conditions. Table S5. Biochemical term enrichment in the exponential phase outlier reactions compared to all metabolic reactions. Table S6. Commonly differentially expressed genes in Liquid, glass beads (APM) and Silt in stationary phase. Table S7. Subset of commonly differentially expressed genes in glass beads (APM) and Silt in stationary phase.Additional file 3: DATA 1. Complete list of differentially expressed genes in APM-1H versus liquid-1H (FDR < 0.05). DATA 2. Enriched GO terms among the significantly differentially downregulated genes in cells growing on glass beads (APM) versus liquid media after 1 h inoculation. DATA 3. Complete list of differentially expressed genes in Sand-1H versus APM-1H (FDR < 0.05). DATA 4. Complete list of differentially expressed genes in Silt -1H versus APM-1H (FDR < 0.05). DATA 5. Complete list of differentially expressed genes in Clay-1H versus APM-1H (FDR < 0.05). DATA 6. Enriched GO terms among the significantly differentially upregulated genes in Sand versus APM after 1 h contact. DATA 7. Enriched GO terms among the significantly differentially expressed upregulated genes in Silt versus glass beads (APM) after 1 h contact. DATA 8. Enriched GO terms among the significantly differentially expressed downregulated genes in Silt versus glass beads (APM) after 1 h contact. DATA 9. Enriched GO terms among the significantly differentially expressed upregulated genes in Clay versus glass beads (APM) after 1 h contact. DATA 9. Enriched GO terms among the significantly differentially expressed downregulated genes in Clay versus glass beads (APM) after 1 h contact. DATA 11. Differentially expressed genes in all natural soils-1H vs controls-1 h (ANOVA. FDR < 0.05). DATA 12. Complete list of differentially expressed genes in Jonction-1H vs APM-1H (FDR < 0.05). DATA 13. Commonly differentially expressed genes in soils and Jonction soils after 1 h contact versus control conditions -1H (ANOVA. FDR < 0.05). DATA 14. Unique differentially expressed genes in Jonction-1H vs APM-1H. DATA 15. Enriched GO terms among the significantly differentially expressed upregulated genes in Jonction versus glass beads (APM) after 1 h contact. DATA 16. Enriched GO terms among the significantly differentially expressed downregulated genes in Jonction versus glass beads (APM) after 1 h contact. DATA 17. Comonly differentially expressed genes in Pseudomonas veronii cells growing exponentialy in Liquid, Sand and Silt vs same material -1H (FDR < 0.05). DATA 18. Enriched GO terms among the significantly differentially expressed upregulated genes in LIQUID-EXPO vs LIQUID-1 h. DATA 19. Enriched GO terms among the significantly differentially expressed downregulated genes in LIQUID-EXPO vs LIQUID-1 h. DATA 20. Enriched GO terms among the significantly differentially expressed upregulated genes in SILT-EXPO vs SILT-1 h. DATA 21. Enriched GO terms among the significantly differentially expressed downregulated genes in SILT-EXPO vs SILT-1 h. DATA 22. Enriched GO terms among the significantly differentially expressed upregulated genes in JONCTION-EXPO vs JONCTION-1 h. DATA 23. Enriched GO terms among the significantly differentially expressed downregulated genes in JONCTION-EXPO vs JONCTION-1 h. DATA 24. Complete list of differentially expressed genes in SILT-EXPO vs SILT-1H (FDR < 0.05). DATA 25. Complete list of differentially expressed genes in Jonction-EXPO vs JN-1H (FDR < 0.05). DATA 26. Commonly differentially express genes in APM and Silt in stationary phase (FDR < 0.05). DATA27. Unique differentially express genes Silt in stationary phase (FDR < 0.05). DATA 28. Unique differentially express genes in APM-STAT vs APM-1H (FDR < 0.05). DATA 29. Enriched GO terms among the significantly differentially expressed upregulated genes in SILT-STAT vs SILT-1 h. DATA 30. Enriched GO terms among the significantly differentially expressed downregulated genes in SILT-STAT vs SILT-1 h. DATA 31. Enriched GO terms among the significantly differentially expressed upregulated genes in AS-STAT vs AS-1 h . DATA 32. Enriched GO terms among the significantly differentially expressed downregulated genes in AS-STAT vs AS-1 h. DATA. Enriched GO terms among the significantly differentially expressed upegulated genes in LIQUID-STAT vs LIQUID-1 h. DATA. Enriched GO terms among the significantly differentially expressed downregulated genes in LIQUID-STAT vs LIQUID-1 h.