Aiming to reduce the gap between in vitro and in vivo environment, a complex culture medium, Plasmax, was introduced recently, which includes nutrients and metabolites with concentrations normally found in human plasma. Herein, to study the influence of this medium on cellular behaviors, we utilized Plasmax to cultivate two cancer cell lines, including one breast cancer cell line, MDA-MB-231BR, and one brain cancer cell line, CRL-1620. Cancer cells were harvested and prepared for transcriptomics and proteomics analyses to assess the discrepancies caused by the different nutritional environments of Plasmax and two commercial media: DMEM, and EMEM. Total RNAs of cells were extracted using mammalian total RNA extract kits and analyzed by next-generation RNA sequencing; proteomics analyses were performed using LC-MS/MS. Gene oncology and pathway analysis were employed to study the affected functions. The cellular invasion and cell death were inhibited in MDA-MB-231BR cell line when cultured in Plasmax compared to DMEM and EMEM, whereas the invasion, migration and protein synthesis of CRL-1620 cell line were activated in Plasmax in relative to both commercial media. The expression changes of some proteins were more significant compared to their corresponding transcripts, indicating that Plasmax has more influence upon regulatory processes of proteins after translation. This work provides complementary information to the original study of Plasmax, aiming to facilitate the selection of appropriate media for in vitro cancer cell studies. Due to the different nutrient compositions and concentrations, the culturing environment of media can greatly affect the cell metabolism ,2,3, andRecently, more attentions have been drawn to the optimization of the nutrient compositions and concentrations of culturing media for cancer cell line cultivation ,13,14,15In this study, we made comparisons between Plasmax and two commercially available media, DMEM and EMEM by investigating the expression changes of transcriptomes and proteomes of cancer cell lines to obtain more comprehensive understandings about how this novel medium can affect the cellular functions. A breast cancer cell line (MDA-MB-231BR), and a brain cancer cell line (CRL-1620) were subject to cultivation in the abovementioned three different culturing media and cell harvest. Total RNAs and proteins were extracted and prepared to evaluate the gene and protein expressions, respectively. Unsupervised principal component analysis (PCA) was applied to assess the expression differences within two cancer cell lines cultured by three media. Gene ontology analysis and protein function annotation were performed utilizing Ingenuity pathway analysis software (QIAGEN IPA) to study the altered cellular functions. Significant up- or down-regulations of proteins that are associated with cell movement, cell death and survival, and protein synthesis were observed.Breast cancer cell line MDA-MB-231BR (231BR) was generously provided by Dr. Paul Lockman . Brain cancer cell line CRL-1620 (CRL), DMEM, EMEM, and Fetal Bovine Serum (FBS) were acquired from American Type Culture Collection . Corning Trypsin EDTA 1× (25% Trypsin/2.21 mM EDTA), MS-grade formic acid (FA), HPLC grade water, methanol, and acetonitrile (ACN) were obtained from Fisher Scientific . Mammalian total RNA extraction kit, sodium hydroxide beads, Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC) and sodium deoxycholate (SDC) were purchased from Sigma-Aldrich . NEBNext rRNA depletion fit (human/mouse/rat), and NEBNext Ultra II Directional RNA library prep kit were obtained from New England Biolabs . Trypsin/Lys-C mix, mass-spectrometry-grade, was purchased from Promega .Plasmax medium was prepared based on the formulation  and follTo keep the stock solutions from being contaminated, the mixing process was performed in a strictly aseptic environment. A bottle of 500 mL Plasmax medium was prepared according to the following steps: First, 5mL of stock solution #1 and #2 were added into 482 mL of EBSS, followed by 0.5 mL of solutions #3 and #8. Then, 1mL of solutions #4 and 0.05 mL of #6 were added. Next, 5 mL of BME-Vitamin, 1.625 mL of L-Glutamine and 12.5 mL of fetal bovine serum (FBS) were also added. After adding all stock solutions and other required compositions, the mixture was shaken for 10 min to help the medium mix evenly. Then, the mixture was pushed through a Nalgene rapid-flow filter  with a sterilizing-grade 0.2 μm SFCA membrane installed to remove any bacteria that potentially involved in the medium solution. Finally, the well-prepared 500 mL Plasmax medium was stored in 4 °C for future use.2 flasks. Cell culture was incubated at 37 °C for 4–8 days and fed every 2–4 days. When the cell confluence reached 90%, cells were washed with a 10mL aliquot of PBS twice and detached by 2.2 mL trypsin-EDTA solution. After incubating at 37 °C for 5 min, 5 mL of fresh medium was added to the cell solution to quench trypsin. The cell suspension solution was then moved into three 175 cm2 flasks for biological triplicate and incubated at 37 °C for about 7 days, until it reached 80% of cell confluence. Cells were washed and detached from the flasks using the abovementioned method. After adding 10 mL of fresh medium, cells were harvested by centrifuging at 500× g for 5 min. Cell pellets were collected and washed twice with PBS to get rid of the extra medium. Part of the fresh cells were taken for RNA extraction. The rest of cell samples were stored in −20 °C for future proteomics analyses.The cancer cell lines were cultured and harvested according to the previously reported procedures ,19. Two Cancer cells were first washed with PBS solution and total RNAs were extracted from the cells using a Mammalian Total RNA Extraction Kit following the procedure provided by the vendor. For each cell line, three biological replicates were prepared. After the extraction, a 5-μL aliquot of total RNA solution was taken to determine the concentration using a Nanodrop 1000 spectrophotometer . Then, RNA quality was determined using RNA Screen Tape . Ribosomal RNA depletion was achieved using a NEB Next rRNA Depletion Kit (Human/Mouse/Rat). RNA fragmentation, double stranded cDNA, and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep following the manufacturer’s procedures. PCR enriched libraries were quantified by Qubit and equimolar indexed libraries were pooled. Pooled libraries were quantitatively checked using software Tapestation 2200  and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 S1 flow cell , paired-end reads (2 × 100 bp).g for 10 min. The supernatant, which contained the extracted cell proteins, was collected. Extracted protein from 231BR and CRL cell lines were then treated by tryptic digestion [The protein extraction was accomplished by following previously reported procedures ,21,22. Tigestion ,25. BrieBased on the protein concentration, the remaining samples were first diluted to make a 0.5% concentration of SDC, then reduced by adding 200 mM dithiothreitol (DTT) and incubated at 60 °C for 45 min. Then, the reduced proteins were alkylated by adding 200 mM iodoacetamide (IAA) followed by incubation at 37.5 °C for 45 min in dark. The alkylation reaction was quenched by adding 200 mM DTT again and incubated at 37.5 °C for 30 min. The pH value of each sample was checked before the addition of enzyme to make sure the high efficiency of enzymatic digestion. The protease trypsin/Lys C ,27, was t test was applied to search for significant changes of protein abundances (p < 0.05), followed by Benjamini-Hochberg (BH) procedure for the multiple testing correction. Data sets of significant proteins from both cell lines were uploaded to Ingenuity Pathway Analysis  for functional annotation and pathway analysis to investigate the cellular function discrepancies correlated with the altered protein expressions.The raw data files generated by LC-MS/MS were processed using software MaxQuant v2.0.2.0  . ProteinProteomics analyses were performed using a Dionex UltiMate 3000 nano LC system  coupled with an LTQ Orbitrap Velos mass spectrometer  through a nano-ESI source. A C18 Acclaim PepMap 100 trapping column  was used to remove salt and impurities. Peptide was separated by a C18 Acclaim PepMap RSLC column . The column compartment temperature was set at 29.5 °C. Mobile phase A contained 2% of ACN and 0.1% of FA, while mobile phase B had 0.1% FA in ACN. A 120 min elution method was utilized for the separation of peptides. The gradient of mobile phase B started at 5% over the first 10 min, then increased to 20% in 55 min, 20% to 30% in 25 min, 30% to 50% in 20min, then ramped up to 80% in 1 min and stayed at 80% for 4 min, then dropped back to 5% in 1 min and stayed for 4 min. Two MS scan events were applied for data acquisition, including a full MS scan with the m/z range of 400–2000 at a resolution of 60,000, and a CID MS/MS scan using normalized collision energy (CE) of 35% and a 10 ms activation time. A data dependent acquisition (DDA) method was employed, which selected the top eight most intense ions from the full scan for the MS/MS analysis.www.serummetabolome.ca, accessed on 11 January 2019) [The concentrations of the nutrients and metabolites of the Plasmax media was based on the freely available resource (ry 2019) . Supportp-values between Plasmax and commercial media were calculated using student’s t test. For 231BR cell lines cultivated in Plasmax, a total of 536 and 265 genes were found as statistically significant (p < 0.05) when compared to this cell line cultivated in DMEM and EMEM, respectively. On the other hand, the CRL cell lines cultivated by Plasmax included 349 and 712 significant genes in relative to DMEM and EMEM, respectively. The Venn diagrams in Next generation RNA sequencing was performed to investigate the alterations of gene expressions among the cells cultured in the different media. Overall, 19973 genes from 231BR, and 20465 from CRL were identified . Based oIn gene ontology analysis, differently expressed transcriptomes were clustered according to their molecular and cellular functions ,32. Compp-values of statistically significant transcriptomes were uploaded to IPA. IPA will search against its database and predict the corresponding upstream regulators and the influence on downstream processes based on the given data. In breast cancer cell line 231BR, as shown by In order to gain more understandings about the impacts on cellular functions due to different transcriptome expressions in Plasmax, the pathway analysis was performed using QIAGEN IPA. The two commercial media were employed as baseline, and the transcriptomics results acquired from cell lines cultivated in Plasmax were compared with the counterparts in DMEM and EMEM, respectively. The logarithm of fold changes and 2+ and calmodulin can activate calcium/calmodulin dependent protein kinase (CAMK) and lead to phosphorylation [For the brain cancer cell line CRL, the transcriptomics discrepancies between Plasmax and DMEM resulted in the inhibition of the calcium signaling pathway. As shown in rylation , then acrylation . It has rylation , suggestrylation . On the rylation .t test was then utilized to search for significant changes of protein abundances between Plasmax and commercial media (p < 0.05). In 231BR cell lines , which also suggests that Plasmax might have more impacts on the expressions of proteomes than transcriptomes during the cell line cultivation.To investigate the different expressions of these proteins from transcriptomics level and the correlation between the proteome and the transcriptome, the By using EMEM as the baseline, the inhibition of integrin signaling pathway of 231BR cell line in Plasmax were also observed. As shown by The PAK2 that mentioned above in integrin signaling pathway is also involved in PAK signaling pathway , which iBased on the significant changes of protein expressions, IPA also predicted the upstream regulators and downstream functions. As depicted by In contrary to the 231BR cell line, the cell movement capability, such as cell invasion and migration of brain cancer cell line CRL were more activated when cultured in Plasmax than both DMEM and EMEM. Additionally, the functions related to protein expressions and synthesis were also stimulated by Plasmax. The abovementioned function alterations are associated with the activation of unfolded protein response (UPR) signaling pathway as shown in UPR is an adaptive mechanism in eukaryotic cells to response to the endoplasmic reticulum (ER) stress of misfolded proteins . The prop < 0.01) of proteins involved in the downstream signaling pathway, including heat shock 70 kDa protein 9 (HSPA9) and calnexin (CANX) were also observed in Plasmax. However, similar as discussed in 231BR cell line, the transcripts of HSPA9 and CANX did not present any significant expression changes, (p > 0.9) suggesting that the expressions of these downstream proteins are not only regulated by their transcripts but also controlled by other regulatory processed after transcriptions.In our work, the chaperon GRP78 of CRL cell line and its corresponding transcript HSPA5 were up-regulated by 41% and 28%, respectively, when cultivated in Plasmax than in DMEM. (inset table of Another important finding of CRL cell line is the overexpression of 14-3-3 family proteins. By binding to the phosphorylated serine or threonine residues of the target proteins , 14-3-3 In this study, we investigated and compared the physiological cell culturing media, Plasmax with two commercially available media DMEM and EMEM through transcriptomics and proteomics. Significant differences of gene and protein expressions were observed in two cancer cell lines cultured by these three media. The gene oncology and pathway analyses of significant transcriptomes suggest alterations in cellular functions. The cell proliferation and invasion of 231BR were inhibited by Plasmax compared to DMEM. However, these functions were more activated than in EMEM. For CRL, the calcium signaling pathway suggested inhibition of cell growth and development in Plasmax in relative to DMEM, while cell cycle was hindered compared to EMEM. On the other hand, the GO analyses of significant proteins indicate that cellular movement, and cell death and survival were the top affected cellular functions in both breast cancer and brain cancer cell lines. According to the expression changes of proteins in 231BR cell line, it also exhibits less cellular invasiveness in Plasmax compared to both commercial media. However, for CRL cell line, its invasion and migration were predicted to be enhanced in Plasmax. The protein synthesis in CRL was another promoted function by Plasmax in relative to DMEM and EMEM. Moreover, the cell death of both cancer cell lines was inhibited when cultivated in Plasmax than both commercial media. The pathway analysis of proteins was utilized to further assess the cellular function alterations introduced by different media. Compared to the commercial media, breast cancer cell line 231BR cultivated in Plasmax exhibited the inhibition of integrin signaling, and PAK signaling pathways, leading to the decrease in cell invasion and cell death, while the activation of unfolded protein response signaling and 14-3-3 mediated signaling pathways in brain cancer cell line CRL are associated with enhanced protein synthesis, cellular migration and invasion.The comparisons between the protein expressions and their corresponding transcriptomes involved in the inhibited or activated pathways suggest that Plasmax has more impacts on these protein expressions, since the RPKM changes of the genes were not as significant as the proteins. In addition, both of transcriptomics and proteomics analyses of 231BR cell line indicated that the invasiveness was inhibited by Plasmax in relative to DMEM. However, the transcriptomics and proteomics analyses suggested opposite results when compared to EMEM. Additionally, the pathway analyses of significant genes and proteins in CRL cell line led to different cellular functions that were affected. The inconsistency between transcriptomics and proteomics indicates that Plasmax might affect the protein expressions after the translations, thus resulting in unique function alterations in proteomics analyses. It can be expected due to the complicated regulation between proteins and their transcripts, with different level of enzymes involved. Overall, these variances of gene and protein expressions indicate the potential influence from the nutrient environment of Plasmax and result in cell behavior changes in different types of cancer cell lines. Our investigation of transcriptomics and proteomics provided complementary information to the original study which focused on the cell proliferation and metabolomics. Researchers may find more clues about media selection for in vitro cell-based transcriptomics or proteomics in future studies.


G12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months.The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model.To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASin vitro sensitivities to KRASG12C inhibitors , but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to modest shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with combinations of MRTX-1257 and the SHP2/PTPN11 inhibitor, RMC-4550. Moreover, treatment with a MRTX-849/RMC-4550 combination yielded transient tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice and durable shrinkage of mKRC.1 tumors. Notably, single-agent MRTX-849 activity in mKRC.1 tumors and the combination response in LLC-NRAS KO tumors was lost when the experiments were performed in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs.The three lines exhibit similar G12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.These new models of murine KRAS G12C inhibitor sotorasib (AMG-510) achieved an objective response rate of 37.1% and the median duration of response was 11.1 months with a comparatively short progression free survival of 6.8 months  state. Distinct from other KRAS codon 12 alleles, KRASG12C retains intrinsic GTP hydrolysis capacity  supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin  at 37°C in a humidified 5% CO67 cells  and Lewiem1LdowKras/J mice  were crossed with B6.129P2-tm1BrnTrp53/J mice (Jackson Labs stock #008462). F1 hybrid mice were genotyped for KRAS and p53 status using PCR on genomic DNA and the following primer pairs: KRASG12C, LSL-Kras Common 5’-TCCAATTCAGTGACTACAGATG-3’, LSL-Kras Mutant 5’-CTAGCCACCATGGCTTGAGT-3’, LSL-Kras Wildtype 5’-ATGTCTTTCCCCAGCACAGT-3’); Trp53, FWD 5’-GGTTAAACCCAGCTTGACCA-3’, REV-5’-GGAGGCAGAGACAGTTGGAG-3’). All PCR reactions employed PCRBIO VeriFi™ mix  according to manufacturer’s protocol with 60°C annealing temperature. Mice heterozygous for KRASG12C and varying p53 backgrounds  were inoculated with Ad-Cre intratracheally and submitted to microCT imaging once monthly until tumor burden was detected. Tumors were harvested, minced, plated on tissue culture plastic and cultured until the stable cell line, mKRC.1, was obtained.B6;129S4-2 plate and cultured for 16 hrs. The NRAS targeting crRNA, ATTO550-labelled tracrRNA and recombinant Cas9 were assembled into ribonucleoprotein (RNP) particles according to IDT manufacturer’s protocol. Assembled RNPs were packaged using Lipofectamine RNAiMAX transfection reagent (ThermoFischer Scientific) according to manufacturer’s instructions, and transfection mixture was added to LLC cells. After a 24-hour incubation, cells were trypsinized, rinsed with 5 mL of PBS, and resuspended into a single-cell suspension in 1 mL of PBS. Single cells were flow sorted into 96-well plates using the MoFlo XDP100 cell sorter for ATTO550 positivity (Beckman Coulter) . For proper compensation of flow cytometry channels, unstained cell lines were used. Single cell clones were expanded, genomic DNA collected, and assessed for NRAS perturbations via PCR and Sanger Sequencing. Two independent clones were identified and indicated as LLC 23 NRAS KO and LLC 46 NRAS KO.A crRNA was designed targeting exon 5 of genomic mouse NRAS using IDT  online software (5’-CACGAACTGGCCAAGAGTTA-3’). LLC cells  were plated in a 35 mmG12C cell lines were generated by the same protocol used for generating LLC NRAS knockout cell lines with the addition of two steps. 1. Prior to transfection, CMT167 cells were incubated with 30 μM HDR Enhancer (IDT) for 1 hour. 2. ssODN’s were added to the transfection mixture and co-transfected into cells along with assembled RNP’s. Single cell subclones from flow sorting were expanded and analyzed for endogenous KRASG12C mutations.A crRNA targeting exon 1 of genomic mouse KRAS (5’-GACTGAGTATAAACTTGTGG-3’) using IDT online software was designed in addition to single stranded donor oligonucleotides (ssODN) homology-directed repair templates for both the positive and negative strands, the sequence for which is: 5’-ATGACTGAGTATAAACTTGTCGTCGTTGGAGCTTGCGGCGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCAGA-3’. CMT167 KRASRNA was submitted to the University of Colorado Sequencing Core where library preparations were generated, and RNA was sequenced on the NovaSeq 6000 to generate 2 x 151 reads. Fastq files were quality checked with FastQC, illumina adapters trimmed with bbduk, and mapped to the mouse mm10 genome with STAR aligner. Counts were generated by STAR’s internal counter and reads were normalized to counts per million (CPM) using the edgeR R package. Differential expression was calculated using the limma R package and the voom function. Heatmaps were generated in Prism 9 . Integrated Genomics Viewer analysis was completed by stripping section of sequence reads from RNAseq dataset and viewing in IGV software .Fastq files were quality checked with FastQC, illumina adapters trimmed with bbduk, and paired reads were mapped to the UCSC mm10 BWA genome with samtools bwa-mem. Output sam files were converted to bam format, sorted, and duplicates were marked with the picard tool, MarkDuplicates. Samtools “mpileup” and bcftools “call” multiallelic caller were used to call variants and generate variant call files (VCF). To identify variants unique to each cell line, bcftools “isec” was used on each cell lines VCF files filtered with bcftools “view” excluding “G/T = 0/0” and including variants with at least a read depth of 10 and a quality score of 20 compared to unfiltered parental VCF files. Variants private to each cell line (001.vcf) were then annotated with ANNOVAR mm10db including refGene, cytoBand, and genomicSuperDups. VCFs were filtered excluding identified “intronic”, and “intergenic” annotated variants under the “Func.refGene tab in addition to “synonymous”, “.”, and “other” under the “ExonicFunc.refGene” tab. Variants were confirmed by interrogating specific regions from the BAM files and uploading those reads to the integrated genomics viewer (IGV). A variant was considered detected reliably if the variant was present in > 10% of the total read depth at the variant position. All code and the parameters used are not included in this report but can be provided upon request.Cells were plated at 100 cells per well in 100 μL in 96-well tissue culture plates and allowed to attach for 24 hrs. Inhibitors were added at various doses as 2X concentrates in 100 μL. After incubation for 7-10 days, cell number per well was assessed using a CyQUANT Direct Cell Proliferation Assay (Invitrogen) according to the manufacturer’s instructions. The data are means and SEM (n=3) and presented as percent of the control values measured in DMSO-treated wells for each cell line.Conditioned media was collected from treated and untreated murine lung cancer cell lines. Chemokine levels were measured using the Invitrogen ELISA Kit  following manufacturer’s instructions. Absorbance was measured at 450 nm. The measured concentration in each sample was normalized to the total cellular protein per dish and the data are presented as pg/μg protein.nu/nu mice were purchased from Envigo . For tumor cell inoculation, parental LLC, LLC 46 NRAS KO, LLC 23 NRAS KO and mKRC.1 cells were grown to 75% confluence, harvested, and resuspended in sterilized phosphate buffered saline. Cells were counted and resuspended to a final concentration of 125,000 cells per 40uL injection. Cell suspensions were directly injected into the left lung of mice through the ribcage. Mice were randomized into groups to receive diluent control, RMC-4550 , MRTX-849 , or the combination by daily oral gavage until primary experimental endpoints were met. Treatment was initiated following confirmation that at least 75% of mice had calculatable primary left lung tumors via microCT imaging . MicroCT imaging was conducted weekly and ITK-SNAP software  and eight-week-old software  was used50 values, the dose-response data were submitted to the “log(inhibitor) vs. normalized response” function with the Prism 9 software. An unpaired Student’s t test (two-tail) was used to determine statistical significance, unless otherwise noted. The P values are denoted by *(P < 0.05), **(P < 0.01), ***(P < 0.001), and ****(P < 0.0001) and were corrected for multiple comparisons (Dunnett). Drug synergy with combinations of KRAS-G12C inhibitors and SHP2 inhibitors was determined with Combenefit, a free software tool for visualization, analysis and quantification of drug combination effects. Data from drug combination assays was processed using the HSA synergy model.Statistical Analysis: Prism 9  was used to perform specific statistical analyses noted in the figure legends. Data are presented as the mean and standard error of the mean (SEM) as indicated. To calculate ICG12C-driven LUAD cell lines to permit investigation of KRASG12C inhibitor responses in immune-competent hosts.The literature supports a role for innate and adaptive immunity in the overall tumoral responses to oncogene-specific inhibitors , 27, 28.G12C mutation  targeting exon 5 of the murine NRAS gene was introduced into parental LLC as a complex with Cas9 and single-cell transfectants were isolated and screened for functional NRAS knockout. Genetic NRAS knockout (KO) in 2 independent LLC clones (LLC 23 NRAS KO and LLC 46 NRAS KO) was validated by RNAseq showing a large deletion near the gRNA targeting region of exon 5 of NRAS (Lewis lung carcinoma (LLC) are derived from a spontaneous lung tumor that arose in a C57BL/6 mouse  and has mutation . Variantmutation  verifiedG12V mutation. CMT167 cells express wild-type Trp53. CRISPR-Cas9-mediated, homology-directed repair was deployed to edit the endogenous G12V mutation to a G12C mutation. Ribonucleoprotein (RNP) particles containing Cas9, a single-stranded oligonucleotide DNA template and a gRNA targeting a sequence within exon 2 of murine KRAS were prepared and transfected into parental CMT cells. Following single cell flow sorting, clones were submitted to functional screening assessed for acquired sensitivity to KRASG12C inhibitors. Putative positive clones that successfully underwent homology-directed repair to contain the KRASG12C mutation were submitted to RNAseq and expression of the G12C allele was verified. RNA sequencing and IGV analysis confirmed an indel in one allele of murine KRAS and a successful recombination containing both KRASG12C as well as the predicted wobble position switches engineered to prevent Cas9 re-cutting of successfully recombined KRASG12C gene  and crossed with Trp53fl/fl mice . The resulting KRASG12C/+; Trp53fl/fl mice were identified by genotyping and submitted to intratracheal administration of Ad-Cre as previously described . Knockout of NRAS increased the sensitivity of LLC NRAS KO cell lines to both KRASG12C inhibitors  compared to parental LLC cells  and indicate that combination therapy with MRTX-849 will be required to achieve clinically significant anti-tumor responses. In this regard, the protein tyrosine phosphatase, PTPN11/SHP2, has emerged as a promising upstream target as it functions to dephosphorylate multiple proteins leading to sustained KRAS GTP cycling and activation . The results revealed a modest reduction in the rate of growth by single agent KRAStivation , 40, 41,tivation  and T cetivation , 15. Bottivation –17.G12C inhibition, we plated LLC 46 NRAS KO, CMT-KRAS-G12C.54 and mKRC.1 cell lines in 96-well plates and assessed cell growth in response to a range of concentrations of MRTX-1257 and RMC-4550 alone and in combination. Analysis of the resulting data  or rapid progression after initial shrinkage (mKRC.1) to MRTX-849 as a single agent. Thus, these new murine KRASG12C-driven lung cancer cell lines propagated as orthotopic tumors reflect treatment responses in KRASG12C-positive patients treated with single agent KRASG12C inhibitors  form  form . Unlike ant form . This re and SOS .https://www.ncbi.nlm.nih.gov/geo/,GSE207098.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by University of Colorado Institutional Animal Care and Use Committee.Conception and design: DS, TH, EK, RN, LH. Development of methodology: DS, TH, AL, RN, LH. Acquisition of data: DS, TH, LH. Analysis and interpretation of data: DS, TH, LH. Writing, review, and/or revision of the manuscript: DS, TH, EK, RN, LH. All authors contributed to the article and approved the submitted version.


Numerous studies have been reported on single- and multicolored highly fluorescent carbon nanoparticles (FCNPs) originating from various sources and their potential applications in bioimaging. Herein, multicolored biocompatible carbon nanoparticles (CNPs) unsheathed from date palm fronds were studied. The extracted CNPs were characterized via several microscopic and spectroscopic techniques. The results revealed that the CNPs were crystalline graphitic and hydrophilic in nature with sizes ranging from 4 to 20 nm. The unsheathed CNPs showed exemplary photoluminescent (PL) properties. They also emitted bright blue colors when exposed to ultraviolet (UV) light. Furthermore, in vitro cellular uptake and cell viability in the presence of CNPs were also investigated. The cell viability of human colon cancer (HCT-116) and breast adenocarcinoma (MCF-7) cell lines with aqueous CNPs at different concentrations was assessed by a cell metabolic activity assay (MTT) for 24 and 48 h incubations. The results were combined to generate dose-response curves for the CNPs and evaluate the severity of their toxicity. The CNPs showed adequate fluorescence with high cell viability for in vitro cell imaging. Under the laser-scanning confocal microscope, the CNPs with HCT-116 and MCF-7 cell lines showed multicolor fluorescence emissions, including blue, green, and red colors when excited at 405, 458, and 561 nm, respectively. These results prove that unsheathed CNPs from date palm fronds can be used in diverse biomedical applications because of their low cytotoxicity, adequate fluorescence, eco-friendly nature, and cheap production. Fluorescent carbon nanoparticles (FCNPs) have unique optical and chemical properties compared to previously used organic fluorescence probes. Carbon nanoparticles (CNPs) are emerging green nanoparticles with superior consonance emission capabilities compared to fluorescence semiconductors . There aSynthesized CNPs based on polymers, metals, silica, and semiconductors are known to possess better multiplexity abilities, making them excellent tools for biomedical applications, such as intracellular monitoring. However, their morphology and concentration remain their major drawbacks, leading to higher cytotoxicity effects. In this regard, it is necessary to investigate the balancing of organic CNP morphology and their doped chemical concentration to reduce their cytotoxicity through selectivity and sensitivity . In addiThe synthesis of CNPs from natural sources using green biosynthesis methods could be better for biomedical applications. The synthesis of CNPs based on trees as a carbon source as an alternative to chemical carbon sources could be environmentally and economically beneficial. Thus, in this work, we synthesized CNPs from date palm fronds. Millions of date palm trees are available worldwide. Date palm fronds are organic and represent a renewable resource, but these trees are burnt as waste for various reasons; therefore, utilizing them as a propitious bio-source for various biomedical applications would be beneficial. Furthermore, the biomass of date palm fronds was found to be rich in lignin content compared with the biomass of other fruits, making them an ideal biomass for carbon extraction. The biomasses extracted from date palm fronds are biocompatible with potential applications in bioimaging and theragnostics ,13. The In this report, novel CNPs with sizes of less than 10 nm were extracted from date palm fronds. The cell viability of the cell lines HCT-116 and MCF-7 in the presence of CNPs was also studied for potential applications in bioimaging. To the best of our knowledge, no study has reported the cell viability and dose-response curve under the application of high concentrations of CNPs to assess their biocompatibility. We also reported the bioimaging potential of crystalline graphitic CNPs in HCT-116 and MCF-7 cell lines through their fluorescence properties. Further investigations should explore the potential applications of these CNPs for cancer diagnosis, such as in multiphoton endomicroscopy , and tre3 with filter caps were purchased from SPL Fife-Sciences, Pocheaon, South Korea. CytoOne 96-well plates (Microtiter plates) with covering lids for cell culture were purchased from Scientific Incorporation, Ocala, FL, USA.Date palm fronds (Phoenix dactylifera) were collected from date farms in Madinah, Saudi Arabia. Thiazolyl blue tetrazolium bromide (MTT) dye, trypsin-EDTA (0.25%), fetal bovine serum (FBS) growth media, phosphate-buffered saline (PBS) solution (pH 7.4), penicillin–streptomycin (Pen/Strep) antibiotic solution, dimethyl sulfoxide (DMSO) media solvent, and high-glucose Dulbecco’s Modified Eagle Medium (DMEM) containing phenol red and L-glutamine were obtained from Invitrogen . SPL tissue culture flasks of sizes 75 and 25 cmAs illustrated in The characterizations of CNPs were performed at the Center of Nanotechnology, Jeddah, Saudi Arabia. The topographies of CNPs were determined using a scanning electron microscope (SEM) model JSM-7600F  and a high-resolution transmission electron microscope (HR-TEM) model JEM 2100F . The XRD was carried out using an Ultima IV model  at room temperature in continuous mode. It emitted graphite-monochromatized Cu-Kα radiation with a wavelength of λ = 1.54 Å. The applied current was 40 mA, and the applied voltage was 40 kV. The scanning axis for XRD patterns at 2θ had an angular range of 10 to 80°, and the first step was 0.05°. FTIR was used to trace the functional group present in the CNPs by applying the attenuated total reflection (ATR) sampling technique using a Nicolet iS10 model . To record the photoluminescence emissions of the CNPs we used a spectral fluorophotometer model RF-5301 . Absorption spectra of the CNPs were recorded with an ultraviolet–visible spectroscope model Lamda 750 . The zeta potential of the CNPs was measured to determine the charges on the CNPs and their colloidal nature with cells using a Malvern particle size analyzer model v2.3 .We estimated the carbonization by investigating the chemical elements using energy dispersive X-ray (EDX) spectroscopy, as shown in 3 with lids were used for culturing cell lines. To maintain the cell growth, 10% (v/v) FBS in DMEM, as well as 1% Pen-Strep solution, was added. The culture flask was incubated under a humidified atmosphere at 37 °C, maintaining 5% CO2. The culture media were replaced when cells attained a high confluence of 90%. Finally, cell harvesting was performed using trypsin solution.The malignant cells, namely the HCT-116 and MCF-7 cell lines, were obtained from the Regenerative Medicine Unit, King Fahad Medical Research Center, Jeddah, Saudi Arabia, where the biological experiments of this study were conducted. Originally, the Regenerative Medicine Unit purchased the cell lines from the American Type Culture Collection . Cell culture flasks of 75 cmFollowing cell harvesting, the cells were seeded using microtiter plates containing ~5000 cells/well. These plates were supplemented with cell growth media and incubated for 24 h under the incubation conditions mentioned above, along with 95% air for adherence. Furthermore, CNPs were prepared at different concentrations , with a CNP concentration of 0 as the control. An MTT assay kit was used to estimate cell viability at time intervals of 24 and 48 h. The principal reaction of the MTT assay is based on the catalyzed conversion of soluble MTT tetrazolium into insoluble crystals of formazan, which are purple in color and produced by the succinate dehydrogenase within mitochondria. The MTT assay was conducted according to the manufacturer’s guidelines.2, maintaining a humid atmosphere. Later, 25 µL of media was removed from each well to dissolve formazan in 50 µL of DMSO [The MTT solution was prepared by dissolving 5 mg/mL of PBS in MTT powder; each well was supplemented with 10 µL of the prepared MTT solution, followed by a further 4 h of incubation at 37 °C with 5% CO of DMSO , mixed w of DMSO . The cel of DMSO :(1)CV%=AData obtained from the cell viability test were applied to plot the dose-response curve for cell lines HCT116 and MCF7 using the logarithmic function; the curve was plotted using cell viability % values versus the different concentrations of aqueous CNPs applied. The estimation of the CNP concentration doses applied to the cell lines was presented in the dose-response curve. The curves for EC20, EC50, EC80, and EC90 showed that these concentrations of CNPs caused the cell viability count to be 20, 50, 80, and 90%, respectively. A non-linear four-parameter logistic (4PL) sigmoid curve (Hill equation) was used to fit the dose-response curve. The curve was fit using the equation provided below .(2)y=A12 cell flask containing media. The detaching process was performed by applying 0.25% trypsin EDTA when a high cell confluency was reached. Then, cells were suspended in DMEM-based media before collection onto the slide. Ten microliters of cells was dropped onto the slides and left for 2 h. Finally, the slides containing the cells were placed into a petri dish and incubated under the abovementioned conditions before imaging with and without aqueous CNPs. Images of cells were captured using a high-resolution scanning confocal laser microscope, model LSM 780, from Carl Zeiss, Aalen, Germany; the laser system was controlled digitally using ZEN 2010 software.The in vitro imaging of cell lines HCT-116 and MCF-7 was carried out using a laser-scanning confocal microscope. The cell lines were cultured in a 25 cmt-test, resulting in a value of p > 0.05. All statistical analyses were performed through Origin version 2022b .Standard deviations (SDs) were taken as the average value of the input data. The significance of the statistical analysis was evaluated using the Student’s −1, which could be assigned to the stretching vibrations of C-O, C-O-C, C=C, and C=O [−1 could be assigned to the O=C=O stretching vibrations; this band was reported as background carbon dioxide CO2 [2 in porous CNPs [−1 could be assigned to the stretching vibration of C-H, while the sharp band at 3400 cm−1 was attributed to vibrations of C-OH [The CNPs showed homogeneous distribution; they appeared to agglomerate as balls, as shown in the SEM image in  and C=O ,27,28,29xide CO2 ,31,32 orous CNPs . The ext of C-OH ,34. The  of C-OH , which wAs was found in the results of the physiochemical properties of the CNPs presented in The CNP dye at a 16 mg/mL concentration diluted in deionized water (DI water) to different concentrations  is presented in The cell viability of the aqueous CNPs was assessed for different concentrations  of CNPs. In this study, CNPs were applied to two famous carcinoma cell lines, namely HCT-116 and MCF-7. The CNPs were dispersed with MCF-7 and HCT-116 cell lines in a 96-well microtiter cell plate. Each cell line was cultured in three wells to obtain triplicate results, with each concentration applied. The average absorbance value of the three wells was used to determine the cell viability, which was calculated using Equation (1). Dose-response curves were generated using the experimentally obtained values of cell viability % at 24 and 48 h of incubation versus the logarithmic concentrations of CNPs for both cell lines. The curves were fitted using the fitting equation. EC50 represents 50% cell viability in response to the applied concentration, so the sigmoid curve at EC50 was denoted as the Hill slope. The dose-response curves for the HCT-116 and MCf-7 cell lines demonstrated a decreasing order of cell viability with corresponding concentrations, as observed from the EC90, EC80 EC50, and EC20 values. The values for EC90, EC80, EC50, EC20, and R2 are summarized in Unsheathed aqueous CNPs from date palm fronds were evaluated for biocompatibility via an MTT colorimetric assay. Both carcinoma cell lines (HCT-116 and MCF-7) were incubated for 24 and 48 h with high CNP doses. The cell viability of the two cell lines was very high, with a survival rate greater than 95% at a 0.5 mg/mL concentration for both incubation times, as depicted in The CNPs reported in this study contained oxygen as the only surface functional group. These CNPs were implemented for bioimaging using a laser-scanning microscope at wavelengths of 405, 458, and 561 nm and emitted mainly blue, green, and red colors, respectively, as depicted in The in vitro cellular uptake and the interaction of CNPs with individual HCT-116 and MCF-7 cells were imaged. A few drops of CNPs at a safer concentration dose of 0.5 mg/mL were applied to the cells. A laser-scanning microscope is used for cell imaging in DAPI mode. The samples were exposed at different excitation wavelengths, including 405, 458, and 561 nm. The images of both cell lines revealed that the CNPs have potential for bioimaging, since they had a zeta potential value of −24.10 (cationic) and a pH of 7.2, demonstrating their ability to penetrate into the cell membrane ,48,49,50The CNPs investigated in this study are environment friendly and originate from date palm trees, with the potential to replace expensive toxic, metallic, and chemical fluorescent dyes for bioimaging. In this study, crystalline graphitic CNPs extracted from date palm fronds demonstrated exemplary PL properties at a pH of 7.23 for bioimaging. Investigations of the CNPs’ fluorescence lifetime, quantum yield at different wavelengths, and fluorescence mechanism need to be conducted in future studies. The immense bioimaging potential and biocompatibility of the CNPs were assessed by an MTT assay with two cell lines, HCT-116 and MCF-7. The adopted concentrations were high compared with those used in most published works due to CNPs’ organic nature. A lower toxicity was recorded under the effect of the CNP concentration of 0.5 mg/mL. The cell viability recorded at 24 h of incubation was 115.86 and 96.47% for HCT-116 and MCF-7 cell lines, respectively, while that at 48 h of incubation was 101.38 and 95.97%, respectively. Both cell lines showed higher cell viability at 48 h of incubation. Both the cell lines were stained with CNPs and then imaged via a fluorescence laser-scanning confocal microscope, which proved that the cell organelles were labeled. However, further studies on efficient strategies for the surface functionalization of CNPs are required for biolabeling, in vivo bioimaging, and various other biomedical applications.


Triatoma and Rhodnius transmit Trypanosoma cruzi, the causative agent of Chagas disease in humans in tropical Latin America. We have developed new cell lines from two of the major vectors of T. cruzi, Triatoma infestans and Rhodnius prolixus, and have shown that they do not harbour any contaminating bacteria or known insect-borne viruses. We propose that the new cell lines may be suitable host cells for Triatoma virus, a natural pathogen of T. infestans that has been proposed as a biocontrol agent but cannot be grown in existing cell lines. The new cell lines may also be used to study interactions between trypanosomes and cells of their vectors. Arthropod vector cell lines play an important role in research on the biology and control of blood-feeding insects and the diseases they transmit. Kissing bugs of the genera Triatoma and Rhodnius are vectors of Chagas disease, a neglected tropical disease of humans in South America caused by Trypanosoma cruzi. Triatoma virus (TrV), a natural pathogen of Triatoma infestans, has been proposed as a possible tool for the bio-control of triatomine bugs, but research into this virus has been hampered by a lack of suitable host cells for in vitro propagation. Here we report establishment and partial characterisation of continuous cell lines from embryos of T. infestans (TIE/LULS54) and Rhodnius prolixus (RPE/LULS53 and RPE/LULS57). RNAseq screening by a sequence-independent, single primer amplification approach confirmed the absence of TrV and other RNA viruses known to infect R. prolixus, indicating that these new cell lines could be used for propagation of TrV. Triatomine bugs of the genera  Trypanosoma cruzi. T. cruzi is the causative agent of Chagas disease, a zoonosis widespread in Latin America [Triatomine bugs of the order Hemiptera, family Reduviidae, also known as kissing bugs, are vectors of the kinetoplastid protozoan parasite  America . Followi America , indicatTriatoma infestans [T. infestans increases their T. cruzi infection rate [T. infestans in Argentina [Rhodnius prolixus [in vitro system for laboratory propagation of the virus outside the insect host [Triatoma or Rhodnius spp. were available to test.Triatoma virus (TrV) , a membenfestans  and therion rate . TrV infrgentina  and in srgentina ,8. Interect host . To dateect host , possiblect host , and no Triatoma maculatum embryo-derived cell cultures surviving for up to 49 days were reported over 50 years ago [T. infestans [T. cruzi [Nosema disstria [Rhodnius.Primary ears ago , and a cnfestans . This ceT. cruzi  and contdisstria . HoweverT. infestans and R. prolixus obtained from laboratory colonies. Screening by a sequence-independent, single primer amplification (SISPA) approach [R. prolixus [Here we report the establishment and partial characterisation of three continuous cell lines derived from embryos of approach  revealedprolixus .T. infestans eggs were harvested from a laboratory colony maintained at LSHTM since 2009. The colony originated from T. infestans insects collected in Paraguay by Dr Nidia Acosta, Universidad Nacional de Asunción—UNA, Paraguay. The T. infestans insects were maintained in glass jars measuring 9.5 cm × 14 cm at 26 °C ± 2 °C, at 70% relative humidity with a 12 h light:12 h dark cycle, and fed every 6–8 weeks on defibrinated horse blood  using a Hemotek® membrane feeding system with 5 mL reservoirs . R. prolixus eggs were harvested from a laboratory colony maintained at Swansea University [R. prolixus insects were fed via a membrane feeding system on human blood. Whole blood was collected by phlebotomists at the School of Medicine, Swansea University, from anonymous healthy adult volunteers by venipuncture into vacuette® tubes containing CPDA . All human blood sampling procedures were reviewed and approved by the South West Wales Research Ethics Committee, REC ref 13/WA/0910. For both insect species, the eggs were used to generate primary cell cultures when the eyes of the majority of developing embryos were visible as reddish spots inside the eggshells.iversity . This coiversity . The R. Three complete culture media were used: L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth, 20% foetal bovine serum (FBS), 2 mM L-glutamine (L-glut), and 100 units/mL of penicillin and 100 µg/mL of streptomycin (antibiotics) (L-15); Hanks balanced salt solution (HBSS) supplemented with 0.5% lactalbumin hydrolysate, 20% FBS, L-glut and antibiotics (H-Lac); and a 1:1 mixture of L-15 and H-Lac (L-15/H-Lac). Complete media were prepared freshly each week. All ingredients were sourced from Invitrogen  or Sigma-Aldrich . T. infestans and R. prolixus eggs were surface-sterilised by immersion in 0.1% benzalkonium chloride for 1–3 min, 70% ethanol for 1 min, sterile ultrapure water for 10 s and HBSS for 10 s in a 30 mm plastic petri dish. The eggs were immersed in fresh HBSS and dissected aseptically by carefully removing the operculum of each egg and gently squeezing out the embryo using sterile watchmakers’ forceps under a binocular dissecting microscope in a horizontal laminar flow cabinet. Embryos with pale or red eye spots were selected for further processing; older embryos with black eye spots (both species) and/or developing reddish exoskeleton (R. prolixus) were rejected. The embryos were collected in a drop of HBSS in a second petri dish and macerated using the watchmakers’ forceps. The macerated tissues were diluted with 1 mL of HBSS and centrifuged at 200× g for 5 min. The tissue pellet was resuspended in 0.5 mL of a solution of 500 µg/mL trypsin  in PBS and incubated at 37 °C for 5 min (R. prolixus) or 10 min (T. infestans). Then 1 mL of complete culture medium was added to inactivate the trypsin and the tissue suspension was centrifuged as before. The tissue pellet was resuspended in 2.2 mL of complete culture medium and placed in a flat-sided culture tube . The sealed tubes were incubated in ambient air at 28 °C. Medium was changed weekly by removal and replacement of ¾ of the total volume. Cultures were monitored for growth by weekly inverted microscope examination. When significant cell multiplication became evident, subcultures were initiated. An equal volume of fresh complete medium (2.2–2.4 mL) was added to the culture, adherent cells and tissue clumps were resuspended by pipetting and half of the resultant suspension was transferred to a new daughter tube, leaving the remainder in the parent tube. When sufficient numbers of tubes were available, half of the contents of two tubes were pooled and transferred to a T25 flask with a total of 5.5 mL of medium. The subculture series were maintained thereafter in parallel in tubes and T25 flasks; cells in flask cultures were passaged 1:1 by resuspending the monolayer using a cell scraper instead of pipetting. Occasionally, cells were resuspended and used to prepare Giemsa-stained cytocentrifuge smears as described previously .g for 5 min. The cell pellet was resuspended in ice-cold complete medium, held on ice and an equal volume of ice-cold complete medium with 20% DMSO was added dropwise with swirling. The resultant suspension was immediately dispensed as 1 mL aliquots into ice-cold cryovials and frozen rapidly on dry ice, before being transferred to vapour-phase liquid nitrogen storage within 30 min. The contents of three culture tubes or one T25 flask yielded three cryovials. Cells were resuscitated from liquid nitrogen storage by rapid thawing in a 37 °C water bath, added immediately to 9 mL of complete medium at room temperature, centrifuged at 300× g for 5 min, resuspended in 2.2 mL of complete medium, placed in a flat-sided tube and incubated at 28 °C. The first medium change was carried out after 5–7 days.Cultured cells were cryopreserved using a modification of a previously-described technique , as follT. infestans and R. prolixus cell DNA using forward primer EukA: 5′-AACCTGGTTGATCCTGCCAGT-3′ and reverse primer EukB: 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ [cytB [Ehrlichia ruminantium-infected bovine endothelial cell culture [T. infestans and R. prolixus cultures were screened for contamination with Mycoplasma spp. using two commercial tests, the Mycoalert Mycoplasma Detection Kit  and the PCR Mycoplasma Test Kit , following the manufacturers’ instructions.Genomic DNA was extracted from harvested cells using a DNeasy Blood and Tissue Kit  following the manufacturer’s instructions for tissue cultures and stored at −20 °C. To confirm the species origin, a fragment of the eukaryotic 18S rRNA gene was amplified by a conventional PCR from the CCTAC-3′ . PCR reaCCTAC-3′  and cytB3′ [cytB  genes we3′ [cytB . All PCR culture . PCR pro culture . RandomlTotal RNA was extracted from cells harvested from two flat-sided tubes or one T25 flask using an RNeasy blood and tissue kit  following the manufacturer’s instructions. Following a TurboDNase treatment, the RNA was reverse transcribed using SuperScript IV Reverse Transcriptase  using primer Sol-A, according to the manufacturer’s instructions. Klenow Fragment  was used for second strand synthesis . The cDNhttps://github.com/spond/gb_taxonomy_tools (accessed on 29 January 2022) based on taxids searched by BLAST and visualised with Krona [https://bugseq.com/free (accessed on 29 January 2022) using the NCBI nt database and visualised with Recentrifuge [NanoFilt was used to filter out sequencing readings less than 100 nucleotides and to then trim 30 nucleotides off the end of the read using the --headcrop and --tailcrop parameters . The firSequences obtained from PCR products sequenced successfully in both directions were deposited in GenBank with accession numbers OP123038 (TIE/LULS54) and OP123039 (RPE/LULS53) for the eukaryotic 18S rRNA gene. The SISPA RNAseq datasets obtained for the three cell lines were deposited in the NCBI Sequence Read Archive (SRA) database under BioProject number PRJNA843092.T. infestans primary cultures, one in H-Lac and two in L-15 derived from a single batch of ~90 eggs, were set up in flat-sided tubes and survive at the time of writing (30 months post-initiation). The primary cultures comprised adherent tissue clumps, some clearly identifiable as sections of limbs, and individual cells with a range of phenotypes including round, epithelial-like, fibroblast-like and contracting muscle cells. As time passed, epithelial-like and muscle cells came to predominate in all cultures. Subculture series were initiated from two of the primary cultures, the one in H-Lac and one of the ones in L-15, after 12 and 9 weeks respectively; growth of the H-Lac culture was slow and this series only reached passage 3 after a total of two years in culture. Three The L-15 culture grew more rapidly, reaching passage 2 after a further 6 weeks and giving rise to a continuous cell line, designated TIE/LULS54. At passage 3, cells of this line were transferred to a T25 flask, where they grew almost as well as in the flat-sided tubes and were easier to dislodge for subculture using a cell scraper. At 10 months after initiation, cells at passage 3 were cryopreserved and successfully resuscitated, and DNA was extracted from a passage 1 culture at that time and from a passage 8 culture at 22 months, for species confirmation and microorganism screening. At 20 months after initiation, RNA was extracted from two passage 6 cultures for RNAseq analysis. At the time of writing, the TIE/LULS54 cell line has reached passage 14; subcultures are carried out at 6-week intervals.TIE/LULS54 cultures comprise a mixture of phenotypes a, includR. prolixus primary cultures were initiated from batches of 12–80 eggs; of these, two in L-15/H-Lac and two in L-15 survive at the time of writing (38–40 months after initiation). In the failed primary cultures, the cells and tissue clumps did not attach well to the surface of the culture tube and did not metabolise beyond the first few weeks, as indicated by rising pH of the culture medium. In the successful primary cultures, tissue clumps attached within the first few days and cells began to migrate out from them, forming sheets of epithelial-like cells and small patches of contracting muscle cells. Areas of large epithelial-like cells containing red pigment, visible to the naked eye, were present in some of the cultures; these persisted for more than 20 months in one of the surviving L-15 cultures. In contrast, the areas of muscle cells ceased to contract after 12–18 months. Attached, and occasionally floating, multicellular vesicles appeared in most of the primary cultures after several months, but did not persist to the time of writing. The subculture series were initiated from three of the surviving primary cultures after 10, 13 and 26 months; of these, the first two gave rise to the continuous cell lines described below while the third, in L-15, has reached passage 4.Eleven R. prolixus cell lines to become established, RPE/LULS53, was generated from 30 eggs in L-15. The first subculture was carried out at 10 months after initiation, with the second and third passages following at 5- and 3-month intervals respectively. Cells at passage 4 were transferred to a T25 flask to facilitate subculturing, 19 months after initiation. The first cryopreservation attempt, carried out with passage 4 cells 20 months after initiation, was successful. DNA was extracted from a passage 1 culture at that time, and from a passage 13 culture at 33 months, for species confirmation and microorganism screening; at 32 months after initiation, RNA was extracted from a passage 12 culture for RNAseq analysis. The RPE/LULS53 cells have reached passage 20 at the time of writing.The first of the two R. prolixus cell line, designated RPE/LULS57, was generated from 44 eggs in L-15/H-Lac. The first subculture was carried out after 19 months, passage 3 cells were successfully cryopreserved at 23 months and the cells have reached passage 14 at the time of writing. DNA was extracted from a passage 6 culture at 30 months for species confirmation and microorganism screening, and RNA for RNAseq was extracted at 29 months from pooled cultures at passages 5 and 7.The second R. prolixus cell lines comprise cells that are considerably smaller than those of the T. infestans line TIE/LULS54 and do not exhibit muscle contractions. Several cell types are present, forming a monolayer of spindle-shaped and fibroblast-like cells , the R. prolixus cell lines now grow faster than the T. infestans cell line. Further study would be required to investigate these contrasting characteristics. All three cell lines are grown in relatively simple culture media, for which all ingredients are commercially available, and are incubated in sealed containers in ambient air, making them suitable for use in laboratories with only basic cell culture facilities and therefore making them applicable in a range of settings in countries where T. cruzi and its vectors are endemic.In the present study, we generated continuous cell lines from two medically-important triatomine bug species. The approach used to generate the TIE/LULS43, RPE/LULS53 and RPE/LULS57 cell lines was relatively simple, and based on techniques used previously to generate a range of insect and tick cell lines ,21,37,38T. infestans and R. prolixus are among the four main vectors of T. cruzi in Latin America [T. cruzi with triatomine cells, trypanosomes cultured with T. infestans TI-32 cells underwent a pattern of differentiation similar to that seen in the vector but not seen in axenically-cultured trypanosomes, indicating that the TI-32 cells produced factor(s) influencing parasite development [Trypanosoma grayi, isolated from a Glossina pallipides tsetse fly, that could not be maintained in axenic culture [T. cruzi for mammalian and insect cells was compared using the non-vector Aedes albopictus mosquito cell line C6/36; in all cases the percentage of cells infected was lower in C6/36 cells than in mammalian cells, but the numbers of parasites associated with the infected cells was similar overall [T. cruzi and the new triatomine cell lines TIE/LULS54, RPE/LULS53 and RPE/LULS56. America  and, as elopment . TI-32 c culture ,42. The  overall . It willT. cruzi [Blastocrithidia triatomae, originally isolated from T. infestans in Argentina; interaction with the trypanosomes appeared to kill the TI-32 cells [Trypanosoma rangeli and R. prolixus haemocytes in vivo and ex vivo [R. prolixus cell lines could be exploited to further examine the role of reactive oxygen species in the pathogenicity of T. rangeli for its host. In addition to trypanosomes, triatomine bugs could play a role in the transmission of bacterial pathogens such as Bartonella spp. and Mycobacterium leprae, the causative agent of leprosy [T. infestans and R. prolixus cell lines might prove to be suitable host cells for the in vitro propagation of M. leprae, as reported previously for a tick cell line [T. infestans may also harbour symbiotic intracellular bacteria of the genus Arsenophonus; Candidatus Arsenophonus triatominarum was isolated from adult female T. infestans haemolymph and propagated in a mosquito cell line [Ca. A. triatominarum, these cells could be used as a more biologically-relevant substrate than mosquito cells for in vitro propagation and the study of this symbiont.Triatomine bugs also harbour trypanosome species that are not known to infect humans but may be pathogenic for their insect hosts and potentially modulate infection with T. cruzi ,44. TI-3 ex vivo  suggest  leprosy ,46. Indeell line . T. infeell line . As the Metarhizium anisopliae, which took longer to kill bugs infected with T. cruzi than uninfected bugs [T. cruzi infection in TrV-infected T. infestans [The environmental costs of insecticides and the increase in reports of insecticide resistance among triatomine bug populations across South America ,49,50 unted bugs  indicatenfestans  deservesT. infestans and R. prolixus cell lines will support productive infection with TrV, we have carried out the first step in this journey by screening them for an existing TrV infection.The reported failure to propagate TrV in cell lines derived from non-triatomine insects , and theT. infestans and R. prolixus for TrV [R. prolixus [R. prolixus insects (0.1–0.6%). These authors used a different approach, generation of paired-end Illumina RNAseq libraries and Illumina HiSeq sequencing, which could have influenced the proportions of viral reads detected in their insect samples. Of likely greater significance, however, are the different origins of the insects used in our study and that of Brito and co-workers [R. prolixus came from a laboratory colony which originated in Brazil, presumably from insects collected from the field in Brazil, sometime prior to 1980. Our R. prolixus came from a laboratory colony originated with insects from Venezuela nearly 100 years ago [The Nanopore sequencing platform was applied in preference to the RT-PCR assays used by others to screen laboratory and field populations of  for TrV ,8, as weprolixus . Interesprolixus  in RNA e-workers . Their Rears ago ; it is tT. infestans cell line TIE/LULS54 harboured the least diverse virome of the three cell lines, as determined by RNAseq, with between 0.0001 and 0.0005% of reads assigned to putative viral sequences, indicating the absence of any complete, replication-competent RNA viruses. This finding suggests that TIE/LULS54 cells would be particularly useful not only for the propagation of TrV, but also for more general investigation of insect host cell responses to RNA virus infection, as any response detected following infection of the cells could be confidently attributed to the presence of the test virus.The T. cruzi and other trypanosomes with their vectors at the cellular level. All three cell lines are deposited in, and available from, the Tick Cell Biobank at the University of Liverpool.In conclusion, the three new triatomine cell lines reported here represent research tools with a high potential for application in studies both on bio-control using TrV and on interaction of


EGFR, PIK3CA, and KRAS, did not predict sensitivity . The machine-learning predictions of combinations that were compared with experimentally generated data showed a bias to the highest quartile of Bliss synergy scores (P = 0.0243). We confirmed feasibility of running such assays on 16 patient samples of freshly isolated NSCLC cells from pleural effusions. We have provided proof of concept for novel methods of using acute ex vivo exposure of cancer cells to targeted anticancer drugs to predict response as single agents or combinations. These approaches could complement current approaches using gene mutations/amplifications/rearrangements as biomarkers and demonstrate the utility of proteomics data to inform treatment selection in the clinic.We hypothesize that the study of acute protein perturbation in signal transduction by targeted anticancer drugs can predict drug sensitivity of these agents used as single agents and rational combination therapy. We assayed dynamic changes in 52 phosphoproteins caused by an acute exposure (1 hour) to clinically relevant concentrations of seven targeted anticancer drugs in 35 non–small cell lung cancer (NSCLC) cell lines and 16 samples of NSCLC cells isolated from pleural effusions. We studied drug sensitivities across 35 cell lines and synergy of combinations of all drugs in six cell lines (252 combinations). We developed orthogonal machine-learning approaches to predict drug response and rational combination therapy. Our methods predicted the most and least sensitive quartiles of drug sensitivity with an AUC of 0.79 and 0.78, respectively, whereas predictions based on mutations in three genes commonly known to predict response to the drug studied, for example,  EGFR  is the leading cause of cancer-related mortality  and is aEGFR , ALK 4)EGFR EGFR , cell transfections (siRNA/CRISPR) or large quantities of protein and extended analysis (mass spectroscopy). These limitations preclude use rapid testing of tumor samples from an individual patient against multiple drugs to enable decision making at any point in their treatment.ex vivo to patient cells. The clinically relevant concentrations and the short exposure of drugs used in these experiments are key to clinical translation of these assays. We establish proof of concept that such an approach is feasible and, in the future, may result in the establishment of platforms that will inform clinical decision making and personalized treatment within 24–48 hours of a biopsy of individual tumors.Here, we quantify dynamic signaling responses within cancer cells to predict drug sensitivity and rational combinations in NSCLC. The approach is applicable both to cancer cell lines and Thirty-five NSCLC cell lines were obtained from the ATCC or from collaborators and STR typed (details in Supplementary Table S1).l-glutamine  and 1x MEM non-essential amino acid solution . Cells were incubated at 37oC with 5% CO2. All cell lines used in experiments were between 4 and 28 passages. Cell lines were tested for Mycoplasma using MycoAlert  within 2 weeks before use.All cell lines were grown in RPMI-1640  except for SK-LU-1 that was grown in DMEM . In addition, all media were supplemented with 10% FBS , 1 mmol/L Cmax) normalized to the protein binding effect in 20% FBS media: Details are provided in the Supplementary Methods.Were obtained from Selleck chemicals. Drug concentrations used for our Luminex assays were based off the clinical maximum plasma concentration (2 tissue culture flasks (Corning Inc.) at 20% FBS until approximately 80% confluent then dosed with one of seven drugs (plus 3 DMSO controls) for 1 hour. Lysate was stored at −80°C until required.Cells were grown in 25 cmMILLIPLEX MAP Akt/mTOR phosphoprotein kit, MILLIPLEX MAPK/SAPK signaling kit, MILLIPLEX MAP RTK phosphoprotein kit  were combined with the following single-plex magnetic bead sets to produce three multiplex Luminex assays: phospho-NFkB, phospho-SRC, phospho-STAT3, phospho-STAT5 A/B, total HSP27 and GAPDH . Bio-Plex Pro phospho-PDGFRa, phospho-PDGFRb and Akt  were combined into a triplex assay. Manufacturer's protocols were followed throughout.Growth inhibition was assessed using 72-hour Sulforhodamine B (SRB) assay (details in Supplementary Methods).Up to 1,000 mL of ascites or pleural fluid was collected by the patient and immunomagentically separated using previously published methods .All patients who had pleural effusions drained for palliative purposes. Pleural fluid was used in the study after investigators has obtained written informed consent. The tissue collection protocols were approved by the Institutional Review Boards and conducted in accordance with the Decleration of Helsinki.To standardize the phosphoproteomic measurements, the control GAPDH measurements were normalized and median-centered, all other data normalized accordingly (see Supplementary Methods).For predictions and feature selection, we created and assessed the performance of a suite of AI-based predictors. First, we used Random Forest recursive feature selection to define the phosphoprotein changes that most contributed to prediction, then trained and validated Random Forest classifiers and regressor functions (details of implementation in Supplementary Methods). Moreover, we in addition used elastic net predictors to predict responses to drugs. Similar models were constructed using notable clinical genomic features of NSCLC to allow comparisons of model performance using the different feature types in predicting drug response.The environmental perturbation score (EPS) is an integrative function across the protein–protein interaction network neighbors. The protein networks were constructed using the highly curated interactome from canSAR . The absU test. Details in Supplementary Methods.Combinations of drugs were assessed using Bliss independence analysis to study synergy. Details in Supplementary Data. The different distribution of the EPS rankings in the highest and lowest quartiles of the combinations ranked by the Bliss independent analysis was tested by a Mann–Whitney Data generated in this study are available upon request from the corresponding author.Cmax adjusted for protein binding in culture medium) of 7 anticancer drugs: Gefitinib (EGFRi), trametinib (MEKi), pictilisib (PI3Ki), capivasertib (AKTi), everolimus (m-TORi), vemurafenib (BRAFi), and luminespib (HSP90i) for 1 hour to recapitulate a clinical setting and eventual translational relevance of our experiments. We chose a limited panel of drugs with well-understood mechanisms of action that had been either licensed or evaluated in clinical trials. We used a panel of 52 relevant phosphoproteins based on the known action of our drug panel and previously validated signal transduction pathways. Using highly curated protein–protein interaction data  and 16 samples of immunomagnetically separated cancer cells from patients with NSCLC with pleural effusions. Cells were exposed to a single concentration  to define the key phosphoprotein changes that predict drug sensitivity in individual cell lines. For comparison, we applied the same algorithms to test the power of known genomic features to predict drug sensitivity. We divided the response data into four quartiles where the first quartile and fourth quartile contain the least and most drug sensitive outcomes, respectively . FeatureEGFR  (Supplementary Table S2). Equally, PIK3CA-mutated cell lines did not show an enrichment for sensitivities to the PI3K inhibitor pictisilib in Quartiles 1 and 2 . Although this may be due to the relatively small sample sizes of numbers of cell lines, these results highlight the limitations of using genotype alone to predict sensitivity to targeted drugs, even those that target a protein that can drive a cancer cell. These experiments were performed before KRAS G12C inhibitors becoming available; however, proteomic analysis outperformed KRAS mutations to predict sensitivity/resistance to all drugs studied. Thus, the predictive power of phosphoproteomic changes in the models studied shows that they could be used to augment current predictive biomarker paradigms based on genotype.In addition, we calculated the predictive performance of each of the three mutated genes when targeted with drugs against their specific protein. Despite EGFR  and PIK3 . The list of EPS values for each node per cell line per drug is presented in the Supplementary Table S3 and an example of proteomic changes caused by capivasertib and trametinib in the HCC827 cell line and the associated EPS score are shown in –D, respein vitro of the 7 drugs in 6 cell lines , resulting in 252 experimentally derived Bliss independence scores. The Bliss independence scores of all the combinations in the 6 cell lines are represented in To test and validate the power of the EPS in predicting synergistic combinations, we conducted blind unbiased pairwise combination screening R2 = 0.0132; Supplementary Fig. S4). However, we observed enrichment of correct predictions in the highest and lowest Bliss data quartiles: Predictions for these quartiles showed significantly skewed distributions . To test the statistical significance of this enrichment, we compared the concordance of our EPS ranking with synergy based on the experimental input data versus 10,000 equivalent rankings based on randomly simulated data (see Supplementary Methods). We found a clear difference between EPS concordance with the experimental data of P values of <0.1 with that of the random rankings  for 1 hour, we exposed immunomagnetically separated cancer cells isolate from fresh pleural effusion aspirates to the 7 drugs under identical conditions. The phosphoprotein analysis was conducted and PCA of phosphoprotein changes due to 7 drugs in established NSCLC cell lines (n = 35) and samples from patients (n = 16) were broadly similar  can be used to predict drug sensitivity: These data were able to outperformed known genetic biomarkers as predictors of sensitivity in the cell line panels and drugs studied. The tailoring of experiments to use clinically relevant concentrations adjusted to protein binding and an acute 1 hour exposure to be used clinically on biopsy specimens in the future, make our proteomic dataset and analysis different from other important recently published work on effects of drugs on proteomic perturbation . HoweverKRAS G12C-driven NSCLC but not colorectal cancer and this is related to feedback loops involving EGFR signaling , xenograft experiments need be done at a scale that is out the scope for academic groups. Showing the results of 1–2 xenograft models to show proof of concept, although conventional, we felt would be against the spirit of unbiased testing and thus we have not conducted these experiments for this article. Such experiments will have to be considered before using the assay in the clinical setting.There are biological complexities such as the role of stroma or the immune system that cannot be captured in the model system described previously in this article. However, we do believe that the current approach is a functional assay that can be delivered in the clinic, which intellectually lies in between genomics (finding mutations/amplifications/deletions or siRNA/CRISPR experiments) and truly phenotypic assays (cell culture/organoid and patient derived xenografts), with the added advantage of being able to near contemporaneously predict sensitivity and synergistic combination therapy. The EPS algorithm, based on acute phosphoproteomic changes, has been validated in To conclude, we have demonstrated for the first time that the use of a focused phosphoproteomic assay and machine learning approaches that has used dynamic phosphorylation in signal transduction to predict sensitivity to drugs and prioritize rational combinations tested on cancer cell lines and patient samples in NSCLC. This is a powerful approach that is orthogonal to genomic markers, is adaptive and individualized, with a clinically meaningful turnaround time. This feasibility study provides proof of concept; however, considerable technical validation is needed before use in patients. If developed further, that this methodology can potentially improve the outcomes of patients with cancer treated with targeted anticancer drugs as a single agent or as combination therapy.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary FigureClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.


Humanised mouse models of GVHD can mimic the clinical setting for GVHD development, with disease progression and tissues impacted like that observed in humans. This review will highlight key findings from preclinical humanised mouse models regarding the role of donor human immune cells, the function of cytokines and cell signalling molecules and their impact on specific target tissues and GVHD development. Further, specific therapeutic strategies tested in these preclinical models reveal key molecular pathways important in reducing the burden of GVHD following allogeneic HSCT.Graft-versus-host disease (GVHD) is a major complication that occurs following allogeneic haematopoietic stem cell transplantation (HSCT) for the treatment of haematological cancers and other blood-related disorders. GVHD is an inflammatory disorder, where the transplanted donor immune cells can mediate an immune response against the recipient and attack host tissues. Despite over 60 years of research, broad-range immune suppression is still used to prevent or treat GVHD, leading to an increased risk of cancer relapse and infection. Therefore, further insights into the disease mechanisms and development of predictive and prognostic biomarkers are key to improving outcomes and reducing GVHD development following allogeneic HSCT. An important preclinical tool to examine the pathophysiology of GVHD and to understand the key mechanisms that lead to GVHD development are preclinical humanised mouse models. Such models of GVHD are now well-established and can provide valuable insights into disease development. This review will focus on models where human peripheral blood mononuclear cells are injected into immune-deficient non-obese diabetic (NOD)- Graft-versus-host disease (GVHD) is an inflammatory disorder that can occur following allogeneic haematopoietic stem cell transplantation (HSCT) ,2. AllogAllogeneic HSCT is a curative therapy for haematological malignancies and other blood-related disorders. This therapy aims to regenerate the immune system following pre-conditioning . In addiDuring GVHD donor immune cells recognise the recipient tissues as foreign and mount an immune mediated attack on host tissues, leading to tissue damage and organ failure . GVHD isWhile several factors may contribute to the severity of GVHD in the recipient, including the level of human leukocyte antigen (HLA) mismatch, pre-conditioning regimes, and the source of donor cells , furtherMechanisms for GVHD progression and the development of treatments for the prevention of this disease have come mainly from allogeneic mouse models, which do not always translate to the clinical setting . HumanisThis article will briefly review current approaches in allogeneic HSCT, before discussing GVHD and humanised mouse models. Finally, this article will detail cellular and molecular mechanisms in GVHD as observed through studies in humanised mouse models using functionally related strains.Allogeneic HSCT is largely used to treat haematological malignancies . This trDespite the life-saving benefits of allogeneic HSCT in people with haematologic malignancies, GVHD occurs in 30–70% of recipients . To mana+ cell depletion), anti-proliferative reagents including mycophenolate mofetil or sirolimus (rapamycin), targeting activation with basiliximab [interleukin (IL)-2 receptor (IL-2R) blockade] or daclizumab (IL-2R blockade), using protease inhibitors like α1-antitrypsin or Janus-associated kinase (JAK) inhibitors, targeting tissue homing molecules including vedolizumab [alpha 4 β 7 (α4β7) integrin blockade] or using extra-corporal photopheresis or faecal microbiota transplantation  and tocilizumab (IL-6R blockade), use of the kinase inhibitor ruxolitinib (JAK 1/2 inhibition) or the anti-proliferative reagent azacitidine (DNA-methyltransferase inhibition), glycoprotein targeting with sitagliptin (CD26/dipeptidyl peptidase-4 inhibition), and regulatory therapies including low dose IL-2 and transfer of Tregs .Use of each new GVHD therapy or combination therapy needs to balance the risk of GVHD prevention with the risk of engraftment failure, infection and in the case of haematological malignancies, disease relapse. To this end humanised mice have provided valuable models in understanding the mechanisms of GVHD, as well as in the testing the efficacy of new GVHD therapies and their impact on GVT immunity.GVHD is still a major issue in allogeneic HSCT recipients, despite the numerous treatment options outlined above, with 30–50% of recipients developing acute GVHD  and 30–7Acute GVHD is mediated by host antigen-presenting cells (APCs) and donor T cells in a three-stage process, defined as the afferent, efferent and effector phases . The aff+ and CD8+ T cells, respectively, recognise host major histocompatibility complex (MHC) class II and I molecules on APCs, with mismatches between host and donor MHC molecules driving the activation of T cells [+ T cells can differentiate into Th1 and Th17 subtypes following T-cell receptor (TCR) stimulation and co-stimulation with cytokines like IL-12 [+ T cells into various subsets with differing cytokine profiles [The efferent phase begins when activated host APCs stimulate donor T cells. Donor CD4 T cells . Moreove T cells . Followi T cells . For exake IL-12  or transke IL-12 , respectprofiles . Regulatprofiles ,46. Brieprofiles .+ and CD8+ T cells directly attack target host cells through the Fas/FasL pathway and perforin/granzyme pathways, respectively [The effector phase occurs when activated alloreactive donor T cells migrate to host tissues, including the gastrointestinal tract, skin, liver and lung, and initiate a severe inflammatory response . T-cell ectively . Furtherectively . For exaectively  and miR1ectively  increaseectively  and miR1ectively  decreaseectively . AltogetChronic GVHD has also been described to occur in three phases . The firDespite current prophylaxis and treatment options, GVHD is a prevalent complication following allogeneic HSCT and remains a major cause of morbidity and mortality. A better understanding of the mechanisms of GVHD pathogenesis is needed to overcome this limitation and improve the success of allogeneic HSCT as a treatment for haematological malignancies. To this end, humanised mice have provided valuable models in understanding the mechanisms of GVHD, as well as the testing of the efficacy of new GVHD therapies and their impact on GVT immunity in relation to haematological malignancies. However, such models are limited mainly to acute GVHD, which will be the focus of the remaining article, with acute GVHD denoted simply as GVHD hereafter.Humanised mouse models can be broadly classified as those that involve the expression of specific human gene products within mice, including instances in which a specific mouse gene is replaced by the human orthologue or those that involve the injection of human cells into immunodeficient mice . The latIl2rg gene [tm1WjlIl2rg) or a truncated IL-2R γ-chain lacking the intracytoplasmic domain (tm1SugIl2rg), both of which prevent murine NK cell development [scid mutation in the gene encoding the DNA-dependent protein kinase catalytic subunit (scidPrkdc) or the targeted tm1Fwa mutation in the recombination-activating gene 2 (tm1FwaRag2), both of which impair murine T- and B-cell development [Most commonly, GVHD is studied by the transplantation of hPBMCs into immunodeficient mice encoding targeted mutations in the 2rg gene , which relopment . The bacelopment . Mice arelopment . Mice enelopment ,71.scidIl2rgtm1WjlPrkdc (NSG) mice the most published. The first detailed report using this strain revealed that intravenous (i.v.) injection with as few as 5 × 106 hPBMCs into irradiated (2 Gy) NSG mice results in lethal GVHD [6 hPBMCs into non-irradiated NSG mice could induce lethal but delayed GVHD. A preliminary observation from the same group indicated that intraperitoneal (i.p.) injection of 20 × 106 hPBMCs into non-irradiated NSG results in human leukocyte engraftment, but details regarding GVHD development in this study were limited [scidIl2rgtm1SugPrkdc (NOG) mice. The i.v. or i.p. injection of at least 1 × 104 or 1 × 107 hPBMCs, respectively, into irradiated (2.5 Gy) NOG mice results in lethal GVHD  mice, with GVHD also able to occur in the absence of irradiation [The most commonly used hPBMC mouse model to study GVHD involves three related strains  with NODhal GVHD . This sa limited . Since thal GVHD , with thNOG mice . The thiadiation  (Table 1W-JTyr+PrkdcscidIl2rgtm1WjlKit (NBSGW) mice [The studies above indicate that the pre-conditioning of mice with irradiation and the subsequent tissue damage is not necessary for the induction of GVHD following hPBMC injection. These findings parallel reports in other models, where it was revealed that i.v. injection of human peripheral blood T cells into NOG mice  or the rGW) mice  could inGW) mice . LikewisGW) mice . Human TGW) mice . Thus, tGW) mice  and NBSGGW) mice  mice, bu6 hPBMCs into irradiated C.Cg-tm1FwaIL2rγtm1SugRag2 (BRG) mice (more commonly referred to as BALB/c-−/−IL2rγ−/−Rag2 mice) and which also leads to lethal GVHD [scid-IL2Rγnull mice. Use of these humanised mouse models has promoted insights into the mechanisms of GVHD  , which can impair autoimmune disease [In NSG mice injected with hPBMCs, Tregs are known to be reduced over time . Therefo of IL-2 , indicat of IL-2 , indirecuce GVHD . Finally8+ Tregs . This ef disease , can alsMDSCs are a heterogeneous population of myeloid precursors with emerging inhibitory roles in preventing GVHD . As for Finally, tolerogenic DCs may also have a role in preventing GVHD. Co-injection of human tolerogenic DCs (induced by active vitamin D) with hPBMCs reduced GVHD and corresponded to an increase in human Tregs . CollectMSCs are multipotent stem cells that can not only differentiate into various cell types but can also regulate the immune response in many diseases including GVHD . As suchex vivo manipulated MSCs prior to injection, reporting decreased GVHD [Numerous clinical trials investigating MSCs as a therapy for GVHD in allogeneic HSCT recipients have been conducted, with the vast majority electing to use allogeneic bone marrow-derived MSCs . These tsed GVHD .Inhibitors and ligands have been examined in a number of humanised mouse studies and have helped elucidate cell signalling mechanisms in GVHD. The majority of such studies have shown the prevention or reduction of GVHD following administration of these compounds, highlighting important roles for DAMPs (and their receptors), co-stimulatory molecules, cytokines (and their receptors) and intracellular pathways in GVHD development  reduced GVHD, which corresponded to a reduction in a number of human pro-inflammatory cytokines including IFN-γ, TNF-α, IL-2, IL-4 and IL-17A in humanised mice . InhibitIn contrast with the co-stimulatory molecules above, mAb blockade of the immune checkpoint molecule programmed cell death (PD) molecule-1  worsened GVHD in humanised mice, which corresponded to an increase in human IFN-γ . This suCytokines, including chemokines, and their respective receptors play a pleiotropic role in cell signalling. These have been indirectly studied in the context of GVHD development and progression with a limited number of studies directly investigating cytokines and their receptors in humanised mice. The latter will be the focus below.+ T cells, corresponding to a reduction in murine neutrophils and macrophages, and reduced C-X-C chemokine ligand (CXCL) 1 and CXCL5 in skin lesions [+ T cells alone could not engraft NOG mice, revealed that the transgenic expression of human IL-2 could support CD8+ T cell engraftment and GVHD development, with the authors suggesting that IL-2 from CD4+ T cells facilitates CD8+ T cell engraftment in this model following injection of hPBMCs. Consistent with this, transgene expression of human IL-2 promotes the expansion of both human CD4+ and CD8+ T cells and GVHD development in NSG mice [+ T cells and limits GVHD development compared with transgene expression of IL-2 alone [Blockade of cytokines or their receptors has revealed the role of signalling axes in the development of GVHD in humanised mice. Fusion protein blockade of the soluble TNF-α receptor delayed progression of GVHD, which corresponded to a reduction in human leukocyte engraftment and was interpreted to be an indicator of GVHD development . Blockad lesions . MoreoveNSG mice . HoweverNSG mice , an effe-2 alone . Paradox-2 alone . Collect+ T cells and decreased Tregs [Two studies have examined the impact of exogenous cytokines on GVHD development in humanised mice. Transgene delivery of human IL-4 was shown to reduce GVHD, which corresponded with a reduction in human T-cell engraftment , suggested Tregs , possiblFinally, blockade of C-C chemokine receptor (CCR) 7 (CD197), which facilitates leukocyte trafficking into secondary lymphoid tissues, reduced GVHD in humanised mice, which corresponded to a reduction in leukocyte engraftment in spleens of these mice . This st+ and CD8+ T cell engraftment and reduced human Treg engraftment [+ cells and increased human Tregs [By extension, the above studies of DAMPs, co-stimulatory molecules and cytokines indirectly implicate a number of intracellular signalling pathways in the development and progression of GVHD. As such, numerous studies have used small molecule inhibitors to investigate the role of various intracellular signalling molecules in GVHD . Blockade of JAK 1, which is downstream of IL-2, IL-4 and IL-6 receptor family members, demonstrates a role for this kinase in GVHD development. Blockade of JAK 1 corresponded to increased human CD4raftment . Dual blraftment . Likewisan Tregs . CollectOther studies of humanised mice indicate that kinases unrelated to the above are also involved in GVHD progression. Blockade of mTOR has demonstrated a role for this kinase in the development of GVHD through production of human IFN-γ and TNF-α and impairment in human Treg numbers . Finally+ and CD8+ T cells in the pathogenesis of GVHD are required, as evidence from current studies is still conflicting. Furthermore, the role of Th17 cells and the mechanisms behind their pathogenicity in a GVHD setting, as well as other Th subsets need to be further explored. Whilst there are numerous studies that investigate the role of CD4+ Tregs in alleviating GVHD, future studies should seek to determine the efficacy of other regulatory cells such as KIR+CD8+ T cells and MDSCs. Further investigation of key points within cell signalling pathways may also elucidate additional mechanisms through which GVHD arises in humanised mice. Outcomes of these studies may provide valuable insights into the best approach to prevent and reduce GVHD and lead to better biomarkers for predicting disease development in a clinical setting. Finally, studies that investigate such mechanisms need to assess the role of these mechanisms in the development of GVT immunity to prevent increased rates of disease relapse.It is evident that humanised mouse models are a useful preclinical tool to examine the mechanisms of GVHD development. Further studies investigating the specific role of CD4