The chemoattractant adipokine chemerin has been found to be elevated in several types of cancer, including colorectal carcinoma. The functional role of chemerin in colorectal carcinoma, however, has not been elucidated to date. This study analyses the impact of the chemerin analog CG34 on proliferation, colony formation, and migration in the human colorectal cancer cell lines HCT116, HT29 and SW620. In addition, the effect of systemic CG34 treatment is investigated in two xenograft mouse models of colorectal cancer (HCT116-luc and HT29-luc). The results of this study suggest there is a stimulatory role of chemerin receptor activation on the growth of colorectal carcinoma.Background: Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation, and migration of CRC cell lines in vitro as well as in animal models. Methods: In vitro, microscopic assays to study proliferation, as well as a scratch-wound assay for migration monitoring, were applied using the human CRC cell lines HCT116, HT29, and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by an ex-vivo analysis of vessel density and mitotic activity. Results: While the proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signals of HCT116-luc and HT29-luc xenografts. Conclusions: The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC. Chemerin is a regulatory adipokine involved in energy metabolism, regulation of immune function, and other physiological processes such as chemotaxis, differentiation, and proliferation. It exerts its action via its G protein-coupled receptors chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C chemokine receptor-like 2 CCRL2 ,4,5,6. CChemerin has also been associated with several functions of tumor progression, such as growth, invasion, metastasis, recruitment of immunocytes, and formation of new vessels ,19. In aRegarding colorectal carcinoma (CRC), chemerin plasma concentration was found to be associated with the risk of incident CRC and to be independent of established CRC risk factors ,28. In aSo far, however, the potential regulation of CRC tumorigenesis and progression has not been investigated in an experimental setting. Recently, our group has reported the development of the metabolically stable high-affinity chemerin peptide agonist CG34 that potently activates both CMKLR1 as well as GPR1. Chelator-linked and radionuclide-loaded CG34 was able to detect via PET/MRI imaging breast cancer in a mouse model that used xenografts of the endogenously CMKLR1-expressing human cell line DU4475 . In this study, the chemerin analog CG34 was used to investigate the possible regulation of three human CRC cell line models in vitro with regard to proliferation, colony formation, and migration. In addition, CG34 was utilized to study the influence of chemerin receptor signaling on growth and vascularization in two human CRC cell xenograft mouse models. The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.2. Cells were passaged every 2–3 days.The luciferase-expressing cell lines HCT116-luc and HT29-luc were purchased from Caliper . The cell line SW620-luc cell line was created by transduction in our laboratory. All other cell lines were purchased from ATCC/LGC Standards  or CLS . The cell lines were cultured in RPMI 1640 medium  or McCoy’s 5A modified medium , each supplemented with 10% fetal calf serum . All tumor cell lines were cultured in an incubator  providing a humidified atmosphere at 37 °C with 5% CO® Probe Master Kit (Roche) additionally contains Taq DNA Polymerase, reaction buffer, and nucleotides . For CyberGreen-based PCR, Sso Fast EvaGreen Supermix  was used. PCR was performed using the CFX96 real-time system (Bio-Rad). For this purpose, 30 ng of cDNA were added. The analysis was performed using qBase PLUS  according to the ∆∆Ct-method. As multiple reference genes were included in the normalization, the geometric mean of the relative housekeeping expression was first determined and then this value was used to normalize the target gene expression. Subsequently, these data were normalized to a cq value of 34. The preparation of RNA for RT-qPCR experiments was performed according to the manufacturer’s instructions using the RNeasy Plus Mini Kit . Isolated RNA was transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit . For this, 3.2 µg of RNA was transcribed into 80 µL of cDNA  according to the manufacturer’s protocol. In addition, as a negative control for each sample, a preparation without reverse transcriptase was analyzed in RT-qPCR. Quantitative real-time human-specific primer sets for TaqMan and CyberGreen amplifications were obtained from Life Technologies , SIGMA , or Roche . Primer sequences, or product numbers, are provided in For the detection of chemerin in cell culture supernatants, ELISA kits from DRG Instruments  were used. Cells were incubated in a full medium at a density of approximately 70% for 24 h; supernatants were immediately used for ELISA or frozen at −80 °C. The ELISA was performed according to the manufacturer’s protocol with the supplied standards. MaxiSorp 96-well plates  were used. The measurement was performed in the SpectramaxPlus384 reader  at 650 nm (before the addition of the stop solution) or 450 nm (after addition of the stop solution) and the concentration was determined based on the standard curve using SoftMax Pro 5.3.After reaching a confluence level of 60–70%, cells were trypsinized  and seeded onto a 96-well plate with transparent, microscopy grade bottom . Cell seeding was then performed by pipetting 100 µL containing 10,000 cells per well. At 1 h after cell seeding, the substances to be tested, including negative controls  were added to the cells in a volume of 100 µL per well. The chemerin analog CG34  was diluted in the medium corresponding to the negative controls, as stated above. To avoid loss of substance activity over time, a medium with or without compound was exchanged every 24 h. After 96 h, cells were fixed for 10 min in a PBS-buffered 4% PFA solution  pipetted directly into the medium. Then, cells were subjected to 10 min of incubation with 5 µg/mL DAPI  and 0.1% Triton  in PBS. Plates were washed 2 times with PBS. After the last wash, PBS was left on the plate. The plate was covered with a transparent foil and stored at 4 °C until measurement. The number of nuclei was determined using high-content analysis with the IN Cell Analyzer automated microscope . Data are reported as relative cell numbers in % of the positive control.After reaching a confluence level of 60–70%, cells were trypsinized and seeded onto a 96-well standard cell culture plate . Cell seeding was then performed done by pipetting 100 µL each with 10,000 cells per well. Directly after cell seeding, 100 µL per well of RPMI1640 with 10% FCS containing the chemerin analog CG34 were added to the cells. As negative controls, other wells were incubated in RPMI1640 with 10% FCS only. Plates were then placed for up to five days into an automated microscopy system . Bright-field images of each well were taken every two hours. To avoid loss of substance activity over time, a medium with or without compound was exchanged every 48 h. Using the Incucyte software, cell confluence was determined by an adapted segmentation algorithm. Using kinetic confluency data, the rate constant k for cell growth up to three days after seeding was calculated using the exponential growth equation (Y = Y0 × exp(k × X) in GraphPad Prism 7.0 . Cells were seeded in a flask and incubated for 3 days until reaching 70% confluence. Cells were then trypsinized, suspended in serum-free RPMI1640 medium, and counted using a Neubauer counting chamber. Four ml of a cell suspension containing 30,000 cells per 1 mL of FCS-free medium were prepared. In parallel, a sterile β-mercaptoethanol solution was prepared ) in 8.45 mL of PBS. In addition, methylcellulose aliquots  were pre-warmed at 37 °C. Under sterile conditions, a mixture of 3.6 mL of methylcellulose, 2.7 mL of FCS, 60 µL of mercaptoethanol solution, 770 µL of Iscove’s medium , and 300 µL of the cell suspension was prepared in a tube. The tube was shaken vigorously for approximately 5 min until the preparation took on a pink color. Then, 1.63 mL of agar previously boiled in the microwave and dissolved in the serum-free medium were added to the batch, shaken vigorously repeatedly, and placed in the cell incubator for 20 min. After the incubation period had elapsed, 1 mL of the resulting cell mixture was spread evenly on a gridded 3.5-cm cell culture dish using an insulin syringe, taking care to prevent the formation of air bubbles. For both CG34 treatments as well as the medium control, three small cell culture dishes  were filled with the cell preparation and placed in a large cell culture dish, which was previously loaded with a small dish filled with PBS to prevent the preparation from drying out. The next day, each cell batch was treated with 100 µL of the substance to be tested for stimulation of colony formation. The negative control consisted of the equivalent treatment with 100 µL of medium containing a concentration of FCS specified for the cell line under investigation (see DAPI assay). CG34 was diluted (100 nM) in the medium of the FCS concentration corresponding to the negative control. Subsequently, compound or negative control additions were made once daily, with only 10 µL added to each batch. The large cell culture dishes containing the preparations were incubated in an incubator for 7 days. On the last day of the experiment, tumor cell colonies were manually counted at a defined size of at least 20 cells per colony under a bright-field microscope at 40× magnification. Data are reported as relative cell numbers in % of the negative control.After reaching a confluence level of 80–90%, cells were trypsinized and seeded in a 96-well standard cell culture plate . Cell seeding was then performed done by pipetting 100 µL of RPMI1640 with 10% FCS with 75,000 cells per well. The next day, cell monolayers were treated with a 96-head scratch tool. Cells were washed once with a medium. Thereafter, 200 µL per well of RPMI1640 with 10% FCS containing the chemerin analog CG34 (10 µM) was added to the cells. As negative controls, other wells were incubated in RPMI1640 with 10% FCS only. Plates were then placed into an automated microscopy system  for up to 24 h. Using the Incucyte software, the % relative change in wound density from 8 to 20 h after compound addition was determined by an adapted segmentation algorithm. 6 cells in 100 µL RPMI1640 each of the HT29-luc (left flank) or HCT116-luc (right flank). Tumor growth was monitored once weekly from the first day of the experiment using both caliper size measurements and bioluminescence imaging . On the seventh day, the 12 animals were randomized into two groups with six animals per group. Treatment was started on day eight of the experiment and terminated on day 28. Treatment was administered in a blinded manner , with one group receiving intraperitoneal injections of 200 µL of CG34 (50 nmol) per mouse once daily and the other group receiving equivalent treatment with 200 µL of water. After measuring the length and width of the tumors with a caliper, volume was calculated according to the formula v = 0.5 × (length × width2). To analyze the potential tumor growth effect of CG34 in vivo, a treatment study was performed. Two luciferase-expressing xenograft mouse models for human colorectal carcinoma were used: HCT116-luc and HT29-luc. 12 female 8–10 weeks old NMRI nude mice were each implanted subcutaneously with 5 × 10For bioluminescence imaging, each mouse received an intraperitoneal weight-adapted injection of D-luciferin (150 µL) at a dose of 150 µg/g mouse 5–10 min before imaging. Mice were then placed under isoflurane anesthesia and transferred to the imager, where bioluminescence signal detection took place. The exposure time was 1 min at the beginning for small tumors and would be reduced to a minimum of 10 s as the tumor size increased during the experiment. The Living Image 3.1 software  was used to count the emitted photons, ultimately determining the photon yield in a region of interest corresponding to the tumor under investigation. In addition, animal weight was monitored twice a week. At the designated experimental endpoint, day 31, animals were anesthetized with isoflurane and euthanized by cervical dislocation. The endpoint tumor volume was determined using a caliper of tumors on sacrificed mice . In addition, the weight of the tumors obtained at autopsy was determined. The excised tumors were frozen in liquid nitrogen and stored at −80 °C. 2O2 solution for 10 min. Furthermore, an avidin-biotin blockade was performed using the avidin-biotin blocking kit . To eliminate nonspecific protein interactions with the primary antibody, sections were blocked with 2% skim milk for 30 min. Primary antibody was dissolved 1:50 in 0.1% BSA in PBS and incubated overnight at 4 °C in a humid chamber. As a negative control, the sections were incubated with only the 0.1% BSA solution instead of the primary antibody. In the second step, the biotinylated rabbit anti-rat secondary antibody (DAKO) was applied. After rinsing the sections with PBS, the avidin-biotin complex  was added for 30 min. Freshly prepared AEC development buffer , AEC solution , 30% H2O2) was pipetted onto the sections. The gradual red staining of mouse endothelial cells was followed under the microscope and stopped after 10 min by adding distilled water. Counterstaining with hematoxylin for 10 min was performed to better assess tissue structure. To determine the number of vessels in the tumors, 3 to 4 field-of-view images were taken per tumor preparation using a bright-field microscope at 200× objective magnification. In each field of view, the red-stained vessels were counted using ImageJ v145.To visualize tumor vascularization, immunohistochemical staining was performed using an antibody directed against the mouse endothelial cell surface molecule CD31 . 10 µm-thick cryosections were fixed with 4% PFA solution for 20 min at room temperature. To block endogenous tissue peroxidases and prevent the nonspecific conversion of the dye AEC added later, sections were incubated with a 0.3% HTo determine the fraction of proliferating tumor cells, immunohistochemical staining was done with an antibody against human Ki67 . 10 µm-thick tumor cryosections were fixed with a 4% PFA solution. Further treatment of the slides was performed according to the protocol already described for CD31 staining. The anti-human Ki67 antibody was applied to the sections at a concentration of 0.0016 mg/mL. The primary antibody was pretreated with a biotinylation reagent  for 20 min. In addition, the primary antibody was mixed with a blocking reagent (DAKO) for 20 min before administration to the cryosections. After incubation overnight at 4 °C, the avidin-biotin complex was applied to the tumor tissue sections for 30 min, which was later developed with AEC. The applied staining reaction was stopped after complete red staining of the proliferating Ki67 containing tumor cells using distilled water. Counterstaining with hematoxylin was used to improve the identification of the non-proliferating tumor cells. Counting of Ki67-labeled cells in 3 to 4 fields of view imaged at 200× magnification was performed using the ImageJ v145. To characterize the expression of the primary molecules involved in chemerin signaling in colorectal carcinoma (CRC) and other cancer cell lines, RT-qPCR was performed on cDNAs from such cells. Five CRC and 35 other cell lines, including a CMKLR1-overexpressing HEK293 cell clone, were tested for mRNA levels of CMKLR1, GPR1, CCRL2, and chemerin (RARRES2). High levels of CMKLR1 were only found in the transfected HEK293 clone and the breast cancer line DU4475; CRC cell lines showed moderate expression of CMKLR1. In contrast, higher GPR1 levels were found in a large number of cell lines, including the CRC cell lines LS174T and SW620 as well as in the neuroendocrine colon tumor cell line LCC18. CCRL2 was highly expressed in individual tumor cell lines from the brain, stomach, pancreas, and breast. The chemerin gene RARRES2 showed high expression in quite a number of tumor cell lines, e.g., from the brain and pancreas. CRC cell lines HT29 and SW620 also demonstrated substantial RARRES2 mRNA expression .To verify whether mRNA expression of RARRES2 correlates with the amount of secreted chemerin, supernatants from 33 cell lines were analyzed after 24 h of culture using a chemerin-specific ELISA. Secreted chemerin levels above 0.5 ng/mL were found in two CRC cell lines  and two glioblastoma cell lines . Most of the tumor cell lines, however, showed low or no levels of secreted chemerin .After having established mRNA levels for chemerin and its receptors as well as secreted amounts of chemerin in culture, the functional relevance of these expression patterns in CRC cells was to be studied. Three CRC cell lines with differential expression properties were chosen for further analysis. HCT116 shows moderate to low expression of CMKLR1, GPR1, and chemerin, HT29 is characterized by low to moderate levels of all three receptors but substantial chemerin secretion, and SW620 expresses higher levels of GPR1, moderate to low levels of the other two receptors, but releases high amounts of chemerin peptide. These three cell lines were investigated for a potential influence of chemerin signaling on three relevant tumor cell characteristics: proliferation, colony formation, and migration. As a continuation in an animal model was anticipated, luciferase-transduced cells  were used in these in vitro experiments instead of the wild-type cell lines. As chemerin is known to be subject to fast proteolytic degradation, the metabolically stabilized chemerin peptide analog CG34, a potent agonist of CMKLR1 and GPR1 , was utiIn a first approach, the impact of the chemerin analog CG34 on the proliferation of the three CRC cell lines was studied. Two independent experimental approaches were taken: a high-content analysis quantitation after DAPI-mediated visualization of cellular nuclei A–D and ap = 0.0118), HT29-luc had increased by 9.7% (p = 0.0124), and SW620-luc were stimulated by 16.1% (p = 0.0045) . HCT116-luc cells showed a proliferation increase of 10.1% ( 0.0045) A.Apart from proliferation, the ability of tumor cells to migrate is a crucial property influencing the course and kinetics of tumor progression. In a scratch-wound assay quantifying relative changes of the wound density, chemerin receptor stimulation by CG34 at 10 µM concentration did not change cell migration in any of the three CRC cell line models versus control conditions B–F.As the in vitro analysis of three CRC cell lines had yielded indications for a potential involvement of chemerin receptor signaling in growth regulation, an in vivo study was designed to address this hypothesis in an animal model. Of the three cell models used in vitro, two were chosen for the animal study: HCT116-luc  and HT29-luc . SW620-luc were excluded, as they endogenously secrete high amounts of the ligand chemerin and any effect of an exogenous chemerin analog may be obscured. As the two CRC cell lines chosen were of human origin, immunodeficient NMRI nu/nu nude mice were applied to generate subcutaneous tumor xenografts of HCT116-luc and HT29-luc cells. Animals were treated by i.p. injection daily, starting from day 8 and ending on day 28 after tumor cell inoculation. After randomization, animals received either a bolus of 50 nmol of the chemerin analog CG34 or water. Both tumor size , as well as tumor bioluminescence  as a measure of both tumor size/cell number as well as tumor cell viability, were monitored on days 1, 7, 15, 21, and 28 of the experiment A.3 versus 189 ± 100 mm3 in the CG34 treatment group (p = 0.0151). For HT29-luc xenografts, volumes in the control group (261 ± 147 mm3) were considerably lower than in the treatment group, though not statistically significant   were found to be statistically significant for HCT116-luc xenografts in the control group 53.9 ± 52.4 mm 0.1906) .8 ± 1.1 × 109 p/s versus 6.0 × 109 ± 5.0 × 109 p/s in the CG34 treatment group (p = 0.0152). For HT29-luc xenografts, differences were also high but, due to high variance, not statistically significant: in the control group, total flux was 3.9 × 109 ± 3.5 × 109 p/s as opposed to 9.3 × 109 ± 5.0 × 109 p/s in the treatment group (p = 0.0649) , started to develop differently from day 15 of the experiment for both CRC cell xenografts. Again, values were higher in the treatments groups than in the control groups. On day 28, differences in bioluminescence were found to be statistically significant for HCT116-luc xenografts: in the control group, values were 6.5 × 10 0.0649) .At the termination of the experiment, the sizes and weights of tumors from sacrificed animals were determined. For both volume and weight, all mean values from tumors in the CG34 treatment group were considerably higher than the control group, even though, due to high variance, the differences lacked statistical significance D,E.To investigate a potential involvement of tumor vascularization in growth differences between the groups in the animal study, tissue cryosections from all tumors were prepared and stained immunohistochemically with a marker antibody for blood vessels (CD31). While vessel numbers per area were not different for HCT116-luc xenografts, in HT29-luc xenografts from the CG34 treatment group, they were higher than in the control group A,C.p = 0.362). In the HT29-luc groups, no significant difference was found As tumor growth is associated with the regulation of cellular proliferation, cryosections were also stained with a marker antibody for mitotic activity (Ki67). In this experiment, the percentage of Ki67-positive cells (mean ± SD) was significantly higher in HCT116-luc xenografts from the treatment group (45.8 ± 9.3) than from the control (38.4 ± 10; Chemerin has been demonstrated to be bound by and to activate two heptahelical plasma membrane receptors, CMKLR1 and GPR1, while a third, CCRL2, binds chemerin and is believed to pass on the ligand to the other receptors . In cancIn contrast to the receptors, the ligand chemerin has been extensively characterized with regard to tumor expression and serum levels ,19. MostSeveral tumor types so far have been identified to feature a direct chemerin action on tumor cell proliferation and tumor growth, which may be either stimulating or inhibiting . In gastSimilar to its action on CRC cell colonies, CG34 clearly stimulated the growth of HCT116-luc as well as HT29-luc xenografts in a subcutaneous nude mouse model, as measured by tumor volume and bioluminescence intensity . StatistThough a correlation of chemerin levels and CRC risk has been established ,28,29, aThe chemerin analog CG34 proved to stimulate colony formation as well as xenograft growth in two mouse models of colorectal carcinoma, with intratumor Ki67 cells increased in one of these models. These results strongly indicate a regulating, stimulatory effect of chemerin receptor signaling in colorectal carcinoma.


While murine studies have yielded important insights into the cellular responses of GVHD, differences between murine and human biology has hindered the translation of novel therapies into the clinic. Recently, the field has expanded the ability to investigate primary human T cell responses through the transplantation of human T cells into immunodeficient mice. These xenogeneic HSCT models benefit from the human T cell receptors, CD4 and CD8 proteins having cross-reactivity to murine MHC in addition to several cytokines and co-stimulatory proteins. This has allowed for the direct assessment of key factors in GVHD pathogenesis to be investigated prior to entering clinical trials. In this review, we will summarize the current state of clinical GVHD research and discuss how xenogeneic HSCT models will aid in advancing the current pipeline of novel GVHD prophylaxis therapies into the clinic.Graft- Donnall Thomas, its use has grown exponentially  activity the donor cells have in controlling malignant disease. While αβ T cells (which will be the main focus of this review) have been the primary focus of many studies, ongoing studies are also exploring the role of NK cells and γδ T cells as donor-lymphocyte infusions (DLI) to treat/prevent relapse post-HSCT   grafts were investigated as a means to eliminate GVHD , 8. Whil3533816) . The appin vivo allo-reactive T cell depletion  class I/II genotyping leading to more precise HLA compatible grafts , the estepletion , 18 haveResearch into the fundamental mechanisms of T cell activation following allogeneic HSCT have also advanced tremendously in the past few decades thanks to the genetic tractability and feasibility of using murine models. Through these means, the field has identified numerous pathways/targets that contribute to the GVH reaction with many currently being studied in clinical trials. Unfortunately though, many of these targets will not translate into the clinic; while a less-than-perfect success rate in clinical trials is to be expected, many of these failures are most likely a result of fundamental differences in murine and human immunology . Thus, tIn this review, we will highlight research investigating GVHD using humanized mouse models and discuss how the growing use of humanized mice have the potential to revolutionize the field. To do this, we will highlight each signal of the three-signal hypothesis of T cell activation  individually and contrast the relative insights each model system  has made toward understanding how each signal impacts the development and pathology of GVHD.scid Il2rgtm1WjlPrkdc/SzJ) mouse in 2005 by Leonard Shultz, it became possible to transplant human immune cells and/or immune-progenitors into these mice to study aspects of human immunology/hematopoiesis  without human cell/tissue transplantation, model systems incorporating human immune cells engrafted into these host strains are now referred to as “human immune system” or “HIS” mice.Since the creation of the NSG mouse, several additional “next-generation” immune-deficient mice capable of humanization have been developed including the NSG-SGM3, MISTRG, NBSGW, NOG, NRG and NSG-HLA-A2 mice –26. Impode novo generation. Peripheral blood-humanized mice (PBL-Hu) are commonly used for these studies for these studies though modern versions also utilize isolated T cells or T cell subsets from primary human HSCT graft tissue including bone marrow, G-CSF mobilized peripheral blood or umbilical cord blood. Additionally, human cancer cell lines and patient-derived cancers (commonly referred to patient-derived-xenograft or PDX models) can be transplanted into immunodeficient mice prior to human graft tissue to examine the GVL effect. In this review, we will describe these transplant-related versions of humanized mice research with the general term of “xenogeneic transplant” model systems.Alternatively, immune-deficient mice can also be used to study the human T cell response to xenogeneic antigens. These studies are often much shorter in duration, with the outcome either death due to GVHD or short/moderate persistence of human T cells that are not Of the three T cell activation signals, signal 1 remains arguably the most important and mandatory for successful activation. The strength of any T cell response is due in part to the diversity of the T cell receptor (TCR) repertoire and their ability to recognize non-self-antigens –29. Unliversus CD8 lineage represent modulatory and cytotoxic functions respectively; and the naïve (CD45RA) versus memory (CD45RO) lineages denoting antigen-inexperienced or -experienced respectively. While there are numerous other sub-populations of T cells , this review will focus on the T cell lineages highlighted above.T cells are broadly divided along two separate lineages; the CD4 In murine models of allogeneic HSCT, two independent groups have shown that murine memory T cells are not able to mediate GVHD –35. Thes+ memory T cells for donor-lymphocyte infusions (DLI) respectively  can model these same T cell responses. Initially, it was unknown whether human TCRs could even recognize murine major histocompatibility complex (MHC) complexes and if they did, if the result be a GVHD-like disease , 42. In b, initiate killing of cells infected with a target antigen and that this interaction can be blocked with a CD8 antibody  shortly after transplantation with very few naïve (CD45RA+) T cells detected  also detect a universal transition to an effector memory phenotype several weeks after transplantation  in GVHD development have shown that host hematopoietic and non-hematopoietic APCs are responsible for alloreactive antigen presentation –60. Thisb) into a B10.BR (H-2k), C3H (H-2k) or BALB/c (H-2d) strain. In the clinic, this occurs when there is a defined HLA mismatch at one or more of the HLA loci (see “The Importance of HLA Matching”). Minor histocompatibility mismatches occur between donor and host despite matching HLA loci and are thought to be caused from variations within individual HLA loci  and from the expression of non-classical HLA peptides. While the T cell reactive antigen in minor histocompatibility mismatches is almost certainly due to variations in the presented peptide structure, it is unclear if the primary antigen in MHC mismatches is the mismatched HLA peptide itself or the unique repertoire of peptides it can present compared to the host genotype.Antigenic stimulation in T cell is generally described by the type of HLA mismatching that occurs between the donor and host. MHC mismatches occur due to a complete mismatch of the HLA allele. In murine models, this is often either a C57BL/6 or BALB.B strain  , it is also much higher than in mice, thought to be around 2E6 different clonotypes  . AssuminI locus) . Interesod graft . This maar cells . If xeno present . While tonotypes .Despite the difficulty in overcoming the diversity of the human T cell clonality, two studies (one in humans and the other in mice) suggest that the field may be able to elucidate xeno-reactive  T cell clones in the future. One study investigated the TCRβ repertoire from 15 different allogeneic HSCT patients with various degrees of HLA mismatching . All patT cell development in the thymus is a carefully orchestrated process to ensure that mature T cells have HLA affinity (positive selection) while minimizing reactivity to host antigens (negative selection). As a result, each individual’s TCR repertoire at any given time reflects their own unique HLA genotype. For example, due to the diversity of alleles and variation within those alleles, other than identical twins, it is unlikely more than 10 to 100 people in the world express the exact same immunopeptidome and thus have the exact same clonal TCR architecture though the presence of public TCRs  still occurs quite frequently –29.This highlights the importance of having high quality HLA-matching for allogeneic HSCT, as even a minor variation in HLA could introduce a multitude of alloreactive antigens capable of being recognized by the donor T cell population. Currently, the standard of care is 8/8 allele matching  with 10/10 matches (which include HLA-DQ) becoming increasingly common . Additio+ and CD8+ T cell clones  would classically be discussed next, there are substantially more basic and clinical studies investigating the role of cytokines in GVHD. As such, this review will follow a similar path as the field and discuss the role signal 3 has on GVHD before signal 2.Cytokines are often deemed accessory to optimal T cell activation, though it is clear that they play important roles in directing and shaping the T cell immune response , 71. TheThe role of conditioning regimens on the outcomes of HSCT have changed drastically since the first bone marrow transplantation . HSCT waThe use of both reduced intensity (RIC) and non-myeloablative conditioning (NMA) regimens has expanded the use of allogeneic HSCT to older patients who otherwise would not of survived MAC and children with non-malignant disease who do not require such intensity from their conditioning regimen . AdditioIn regard to the role of conditioning in GVHD development, there are two metrics that are strongly associated with GVHD development. The first is not directly related to conditioning but involves the mismatching of the donor/recipient HLA (see the Importance of HLA Matching above) in which case mismatched transplants generally receive a harsher conditioning regimen to facilitate engraftment. The second is the degree of host damage which is also associated with a more myeloablative conditioning . This isvs-leukemia (GVL) effect by donor graft tissue is well accepted, do allogeneic HSCTs require conditioning? Conditioning serves two purposes beyond that of killing residual leukemia cells (and non-leukemic cells). The first is to remove the host HSPCs. Both MAC and RIC regimens are sufficient to eliminate the host HSPCs while NMA do not offer complete elimination of host HSPCs (Now that the graft-st HSPCs . As a rest HSPCs . The secst HSPCs . As a rest HSPCs .131 (β/γ emitter), astatine211 (α emitter), yttrium90 (β emitter) or saporin (non-radioactive)  and the CD45-astatine211 or 211^At-BC8-B10 antibody has a phase I/II trial ongoing to determine the optimal dose before allogeneic HSCT in patients with AML/ALL/MDS or mixed-phenotype acute leukemia (NCT03128034). As these targeted/non-genotoxic antibody-based conditioning targets progress, it will be important for the field to monitor how the reduction in host damage affects the GVHD penetrance after transplant.To our knowledge, there are currently six different antibody/antibody-drug-conjugate (ADC) based conditioning regimens in development. All of these candidates use either the immune cell marker CD45 or the HSPC-specific marker CD117 to target immune cells for clearance, sparing non-immune cells from any off-target damage. Of the two CD117 antibody-based conditioning regimens, JSP191 and MGTA-117, JSP191 has progressed the furthest so far. JSP191, formerly known as AMG191 or SR-1, has had extensive pre-clinical studies performed showing its ability to deplete both mouse and human HSPCs in a dose-dependent manner that allows for adoptive transfer of allogeneic HSPCs –79. JSP1oactive) , 81. Of One implication for the growth in targeted/non-genotoxic antibody-based conditioning regimens is that the normal cytokine storm fueled by the release of DAMPs from necrotic and pyroptotic cells will be diminished. While it is assumed these type of conditioning regimens will be better tolerated by the patient and reduce the number of NRM deaths, it is unclear how it will affect the frequency and severity of GVHD. In almost all model systems of allogeneic HSCT, the host is conditioned (by γ-irradiation) prior to transplant. This is both a requirement for a successful HSCT in murine, canine, porcine and non-human primate models and mimics what occurs in the clinic. Interestingly, many studies using the immunodeficient NSG mouse have continued this protocol of conditioning prior to transplant despite no longer being a requirement to study GVHD  Figure , 82.In the studies performing xenogeneic transplantation in non-conditioned NSG mice, GVHD development can still occur even in the absence of host damage/cytokine storm. Thus, these limited number of studies suggests that inflammatory cytokines are not required for GVHD initiation though they undoubtedly influence the frequency, severity and pathology of the disease. For example, by studying which murine cytokines are cross-reactive with their cognate human receptors (in addition to which human cytokines are produced by activated human T cells), we may be able to investigate the role of individual cytokines in influencing disease pathology. A non-exhaustive list of murine cytokines and their cross-reactivity on human cells are highlighted in 1 response while skin GVHD is dominated by a TH17 response , chronic IFNγ signaling results in the exhaustion and depletion of HSPCs progenitor populations –91. Specferation . These eferation . Interesferation –96. Phospatients . The preate GVHD . Additioate GVHD . Thus, iate GVHD . Furtherate GVHD . Despiteate GVHD –101.Despite the idea from non-conditioned xenogeneic transplant studies that cytokines may not be essential for GVHD development, they have been one of the most heavily investigated potential mechanisms for GVHD prevention and treatment. Surprisingly though, there has not yet been a clinical study identifying any of these inflammatory cytokines as biomarkers of the GVH response , 103. ToOverall, antibody and/or cytokine regimens for the treatment and/or prevention of GVHD have been met with mixed results. Cytokine therapies involving IL-1RA, IL-2 or an IL-1 decoy receptor have all failed to show efficacy in large phase III clinical trials –107. AntOne hypothesis as to why antibody-directed therapies have not yet shown promise in the treatment/prevention of GVHD is because GVHD is a multi-faceted disease, influenced by a variety of cytokines secreted after condition and that the blocking of just one pro-inflammatory cytokine isn’t sufficient for efficacy , 116. AsThe primary targets of TKIs used for GVHD are JAK1, JAK2 and BTK . While t1 and IL-6 promoting TH17) with the family of TKI taking advantage of the promiscuous use of shared JAK proteins to suppress a broad number of cytokine signals. One criticism of TKIs though is the inability to modulate which cytokines are affected. For example, STAT3 activation downstream of the IL-6R promotes TH17 differentiation but STAT5 activation is also known to promote Treg development  but most likely are integral components of pathology and the perpetuation of disease. This leads to our last section on the role of co-stimulatory and co-inhibitory ligands on GVHD pathogenesis.Co-stimulatory receptors are generally divided into two superfamilies’ based on their extracellular domains, the immunoglobulin superfamily (IgSF) and the TNF receptor superfamily (TNFRSF). Importantly, their signaling pathways rely on adaptor proteins , MAP kinase signaling  and transcription factor activation that is distinct from both TCR and cytokine signaling pathways (Zap70/PI3K and JAK/STAT respectively) , CD40L (CD154) and ICOS (CD278) have been shown to be either upregulated or maintain a high level of expression on human T cells after xenogeneic transplantation , 129. OfCurrently, the most promising agent for GVHD prevention is Abatacept, a CTLA4 fusion protein currently being used in several clinical trials . In one The only other co-stimulatory protein based GVHD prophylaxis treatment currently under investigation is BMS-986004, a CD40L blocking antibody that is currently in a phase 1/2 open label trial (NCT03605927). The aim of this trial is to determine the safety of intravenous injection of BMS-986004 every two weeks starting on day +13 in conjunction with tacrolimus and sirolimus based GVHD prophylaxis and determine the efficacy in preventing grade II-IV aGVHD. In summary, while co-stimulatory proteins have been well-studied in murine models of HSCT, they have been vastly understudied when it comes their efficacy on human T cells, either in xenogeneic transplant models or the clinic. While the field of TKI for GVHD is highly exciting, these early studies also suggest that harnessing the power of co-stimulatory/inhibitory receptors may be the optimal target for future novel GVHD prophylaxis targets.While murine and clinical investigations into GVHD will remain workhorses in the field, it is clear that humanized mouse models are becoming increasingly utilized and offer a unique model system to directly investigate human T cell biology. Xenogeneic transplantation has already provided us insights into the importance of TCR: MHC interactions, the necessity of pro-inflammatory cytokines and a novel tool to investigate the role of co-stimulatory ligands in mediating GVHD development  were both studied in xenogeneic transplant models before moving into late stage clinical trials , 135. WhAbatacept remains the best example of the role xenogeneic transplantations will have in the future as the CTLA-4-fusion protein was first tested in xenogeneic transplantation models before moving into the clinic, where it has now become a highly promising candidate for preventing GVHD though its role in treating established GVHD remains uncertain , 133. AdTheir remains a plethora of exciting research possibilities that are now feasible thanks to the development of xenogeneic transplant model systems. Their feasibility will only grow as we learn more about the cross-reactivity of specific cytokines and co-stimulatory ligand/receptor pairs. With the rapid increase in single-cell-RNA-sequencing (scRNA-seq) and TCR-sequencing capabilities, researchers will be able to delve deeper into the clonality and unique gene expression patterns associated with human T cell responses post-transplant as well as the importance of KIR typing/mismatches in GVHD and GVL , 72. AddNH drafted the manuscript and performed the literature search. MB and CC revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by NIH/NCATS UL1-TR002373 (NH), the Cormac Pediatric Leukemia Fellowship (NH) and the Stem Cell and Regenerative Medicine Center Fellowship (NH). Additional funding includes NIH/NIAID 75N93021C00004 (MB), NIH/NHLBI U01-HL134764 (MB), DOD W81XWH2010669 (MB), St. Baldrick’s-Stand Up to Cancer Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17 (CC), American Cancer Society Research Scholar grant RSG-18-104-01-LIB (CC), NIH/NCI R01 CA215461 (CC) and the MACC Fund (CC). The St. Baldrick’s Foundation collaborates with Stand Up to Cancer. Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. None of these funding sources had any input in the study design, analysis, manuscript preparation or decision to submit for publication.The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.CC reports honorarium from Nektar Therapeutics and Novartis, who had no input in the study design, analysis, manuscript preparation or decision to submit for publication. MB is a consultant for Taconic Biosciences.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chemotherapy appeared to be a significant advancement in cancer research, with fewer side effects. Methotrexate (MTX) is a widely used anticancer drug with strong activity but serious side effects. Several MTX derivatives have been reported, with modifications at various sites to reduce side effects and increase efficacy. The current study uses FTIR spectroscopy to predict the survival fraction of human malignant glioma U87 (MG-U87) cell lines against MTX derivatives. Together with Parent MTX several aldehydes viz. Benzaldehyde, Chlorobenzaldehyde, 2-Chlorobenzaldehyde, 3-Nitrobenzaldehyde, 5-Chloro-2-hydroxybenz-aldehyde, 2-Hydroxy-5-Nitrobenzaldehyde, 2-Thiocarboxyaldehyde, Trans-2-pentenal, and Glutaraldehyde are treated with MTX to obtain MTX derivatives. The prediction of survival fraction of malignant glioma cells is carried out by Lasso, Elastic net and Soft PLS at different concentration levels of synthesized derivatives, including 400 μM, 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM. The cross-validated prediction error is minimised to optimise spectral wavelength selection and model parameters. It appears that the RMSE computed from test data is significantly varying with the change of models (p = 0.012), with the change of concentrations levels (p  It encompasses several cancer types for example leukemia, gestational trophoblastic disease, lung cancer, breast cancer, osteosarcoma and lymphoma3. Different levels of MTX’s dosages are recommended for different types of diseases. For instance, low dose MTX treats Rheumatoid arthritis and other types of inflammatory diseases4, and high doses MTX is used for intraocular lymphoma5. Hence relevant concentrations of MTX are of importance. Together with the wide applications of MTX, it has some drawbacks as it induces life-threatening side effects on sensitive organs. Specific side effects of MTX include diarrhea, feeling weak, cough, increased chances of infection, reduced counts of white blood cell, Within mouth skin deterioration, liver failure, lung disease, lymphoma, and extreme skin rash6.Methotrexate (MTX) is certainly one of the broadly used antimetabolites in cancer chemotherapy that terminates DNA replication mainly by snooping with thymidylate synthesis pathway7 and is expected to minimizes its side effects. MTX structure has several sites, where synthesized modifications in these sites results in MTX derivatives8. One possible way to measure the MTX and its derivatives’ biological effect is to measure the cancer cell survival fraction. It reflects the cancer cells that have retained their reproductive capacity after applying the cancer therapy9. This is customary presented through the survival curve, which describes the link between the absorbed dose and the fraction of cells that survive.Modification in MTX structure enhances its anticancer activity10. FTIR characterize metabolic difference of cardiotonic steroid family11, The subtoxic doses of gemcitabine, a cancer drug, can be monitored using FTIR spectroscopy12, it can model the metabolism disruption track cancer cells14, it can diagnose arthritis(rheumatoid) through serum15, it can recognize most of the cell types found in melanoma cancer16, it can discriminate the anticancer medication reference to their mode of operation for prostate cancer cells17, and it can provide label-free surveillance of therapeutic medicines that is Busulfan and Methotrexate in human serum18.Cancer medications are believed to interact with cell metabolism by many pathways. For characterization of MTX derivatives Fourier Transformation Infrared (FTIR) spectroscopy is potential candidate which is low in cost and fast in processing, which is sensitive to all kinds of molecules present in cells, hence can provide unique and accurate fingerprints of samples. For example, FTIR has been used to predict the migration of glioma cell lines in vitro19. Soft thresholding PLS (StPLS) is a potential variant adds the influential wavelength selection together with response prediction20. Together with PLS, elastic net (Elnet) and least absolute shrinkage and selection operator (Lasso ) are also in practice for modeling cancer cell functions22. These algorithms implements cyclical coordinate descent for computing the regularization path. These approaches have the inner ability to integrate knowledge from all kinds of molecules found in cells and create a spectral signature specific to a cell group. As a result, using FTIR spectroscopy, StPLS, Lasso, and Elnet can be used to predict the survival fraction of MTX derivatives against human malignant glioma U87 (MG-U87) cell lines. These methods work in two steps: model construction and validation.Chemometric analyses are mostly based on widely used partial least squares (PLS) regression. PLS is being extensively used for drug concentration predication, survival and fitness response predicationIn the current study, FTIR spectroscopy on human malignant glioma U87 (MG-U87) cell lines reveals that MTX derivatives develop a reproducible FTIR spectral signature at various concentrations for methotrexate’s unique inhibition of DNA synthesis.23. These cell lines were used to check the initial response as anticancer potential. Initial response can be checked on any cell lines. All solvents were of analytical grade and dried solvents were used in reaction scheme. All reactions were carried out in inert atmosphere of nitrogen. By using the rotary evaporator R-210 solvents were dried. Thin-layer chromatography analytic was conducted on Baker 250F and Whatman MK6F silica gel plates. By using TLC on silica gel plates reaction products were purified. By using UV lamp chemical reactions were visualized. The melting point of reactant and product was determined. These MTX derivatives were dissolved in dry ethanol in round bottom flask, NaOH and given aldehyde was added and reaction mixture was refluxed until TLC indicated disappearance of aldehyde. After completion of reaction pH was adjusted to 5, precipitate was washed with hexane and dried.In this study together with Parent MTX schiff base MTX derivatives with target compounds several aldehydes viz. Benzaldehyde, Chlorobenzaldehyde, 2-Chlorobenzaldehyde, 3-Nitrobenzaldehyde, 5-Chloro-2-hydroxybenz-aldehyde, 2-Hydroxy-5-Nitrobenzaldehyde, 2-Thiocarboxyaldehyde, Trans-2-pentenal, and Glutaraldehyde are considered. MTX is a commonly used drug for various types of cancer. All the new derivatives were synthesized for the first time in study23. To evaluate the anticancer effect of all micro molar MTX derivatives, the growth inhibition assays on MG-U87 cell lines was observed over different MTX-derivative’s incubation times using incremental doses from 12.5 to 400 μM. The human malignant glioma U87 (MG-U87) cell lines were cultured in Dulbecco’s Modified Eagle’s Medium/F12 in 1:1 concentration, with addition of 4 (200 μL/well) and were allowed to grow for 24 h at 37 U87 cell lines were used to check the initial response as anticancer potential. These cell lines were imported from Merck, US supplier. Initial response can be checked on any cell lines. The established method for identification of the survival fraction of U87 is reportedAttenuated Total Reflection-Fourier Transformation Infrared (ATR-FTIR) spectrometer ALPHA 200488 which covers mid-IR (4000–550 cm24. For this use of baseline correction methods is in practice. For this asymmetric least squares (ALS)25 is considered. ALS is based on least squares algorithm which weights explanatory variables with positive differences. For smoothing FTIR spectra includes linear or non-linear residuals from spectroscopic experiment results in non flat base line of spectra. For statistical modeling and analysis of spectral based data set flat line at zero is required24 is used. For this several levels of these parameters are used. For each level of the MTX derivative’s spectra baseline is corrected and is used for further analysis  and others are based on penalized liner models includes elastic net (Elnet) and least absolute shrinkage and selection operator (Lasso )28. Soft thresholding (PLS1) (StPLS) is a potential variant adds the influential wavelength selection together with response prediction20. In order to improve prediction and interpretability, these methods use variable selection and regularisation. As a result, StPLS, Lasso, and Elnet are used in this study to predict the survival fraction of MTX derivatives against human malignant glioma U87 (MG-U87) cell lines using FTIR spectroscopy.Predicting the survival fraction of MTX’s derivatives through FTIR spectral data results in multivariate data with much much larger number of wavenumbers that is explanatory variables compared to the number of samples. Multivariate methods are considered as potential candidate26 the covariance between response 30, hence motivates for variable selection. For variable selection in PLS soft-thresholding step in the PLS (StPLS) algorithm is introduced20. In each iteration components of StPLS loading-weights are computed as: Scaling: Soft-thresholding: Normalizing: The shrinkage parameter 20.In basic PLS algorithm28 are potential candidates. These model implements the coordinate descent algorithm with certain penalty parameter called An alternative to iterative modeling procedure (PLS based) are penalized models. In this regard elastic net (Elnet) and least absolute shrinkage and selection operator (Lasso )3132 the mixture of both In lassoIn current study 1 MTX parent and 9 MTX derivatives are considered, which include Schiff base derivatives MTX with Benzaledehyde, Chlorobenzaldehyde, 2-Chlorobenzaldehyde, 3-Nitrobenzaldehyde, 5-Chloro-2-hydroxybenz-aldehyde, 2-Hydroxy-5-Nitrobenzaldehyde, 2-Thiocarboxyaldehyde, Trans-2-pentenal, and Glutaraldehyde. At various concentrations, the survival fraction of malignant glioma U87 (MG-U87) cell lines against these MTX derivatives is monitored. These concentrations include 400 μM, 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM. The survival fraction of malignant glioma cells at different concentration level of MTX derivatives is presented in Fig. Te current study aims to predict the survival fraction of malignant glioma cell lines against several concentrations of MTX derivatives through FTIR spectroscopy. The 10 samples of MTX derivatives were subjected to FTIR spectroscopic experiments for this purpose. For the projected mathematical models, the threshold for FTIR spectra should be at 0. For ALS baseline adjustment, we have used algorithms. It is dependent on ALS to select their respective parameters. We also used the objective method for optimum parameter tuning, where combinations of various levels of the parameters are evaluated for the survival fraction by PLS.Cross validation establishes the optimal parameter estimates of ALS. For estimating the survival fraction at different stages of concentration, smoothing parameters entclass1pt{minimaWe use StPLS, Lasso and Elnet for the prediction of the survival fraction of MG-U87 against MTX derivatives at different concentrations. RMSE over the test data is used to measure the prediction performance of considered methods. We have randomly divided samples into test and training data for model building and comparison of the predicted model. Although test samples are used for model comparison or evaluation , training data fits the model. There is random bias. In order to remove this bias, the above data validation and calibration process will be replicated within 100 runs. The distribution of optimal parameter estimates for Lasso, Elnet and StPls obtained against concentration level is shown in Fig. Lasso, Elnet and StPls are designed to predict the survival fraction at each stage of the MTX concentration using the above approximate parameters. The validated prediction capabilities that are RMSE standardised over the test data are calculated in each sprint. The analysis of variance results indicating the significance of concentrations, models and their interactions for explaining the variation in RMSE over test data is presented in Table 33. If there are unrelated components with large eigenvalues, the forecast may be worse. As a result, poor prediction of variable selection methods based on PLS is predicted. Figure Two model streams exist for prediction purposes, one uses iterative loading based method (StPLS) and others are based on penalised linear models (Lasso and Elnet). The sample covariance matrix’s own value structure of the explanatory matrix is then used to describe the model prediction action’s characteristics and the covariance between the main components and the response responseInfluential wavenumbers are chosen by the optimum model defined in Fig. 34. For baseline correction R package ’baseline’35 and for model fitting R packages ’glmnet’ and ’plsVarSel’37 are used.For computations, modeling and figures R software is usedVia FTIR spectroscopy by StPLS, Lasso and Elnet, the study predicts the survival fraction of malignant glioma cells against many concentrations of methotrexate derivatives. ASL works well for base-line correction. The survival fraction prediction capabilities are linked with covariance between the survival fraction and spectrum data. Moreover Influential wavenumbers are picked by Lasso at concentrations of 12.5% and 25%, by StPls at concentrations of 50%, 100% and 400%, and by Elnet at concentrations of 200% along with influential functional compounds.Supplementary Information.


Culture model systems that can recapitulate the anatomy and physiology of reproductive organs, such as three-dimensional (3D) organoid culture systems, limit the cost and welfare concerns associated with a research animal colony and provide alternative approaches to study specific processes in humans and animals. These 3D models facilitate a greater understanding of the physiological role of individual cell types and their interactions than can be accomplished with traditional monolayer culture systems. Furthermore, 3D culture systems allow for the examination of specific cellular, molecular, or hormonal interactions, without confounding factors that occur with in vivo models, and provide a powerful approach to study physiological and pathological reproductive conditions. The goal of this paper is to review and compare organoid culture systems to other in vitro cell culture models, currently used to study female reproductive physiology, with an emphasis on the role of extracellular vesicle interactions. The critical role of extracellular vesicles for intercellular communication in physiological processes, including reproduction, has been well documented, and an overview of the roles of extracellular vesicles in organoid systems will be provided. Finally, we will propose future directions for understanding the role of extracellular vesicles in normal and pathological conditions of reproductive organs, utilizing 3D organoid culture systems. Reproductive physiology involves extensive communication between different organs and tissues and between different cells within tissues. A variety of models have been developed to study cellular communication in reproductive physiology. However, developing physiologically relevant in vitro models has been challenging because these dynamic models require specific cell targeting with a functional response to stimuli in a controlled environment. Therefore, the development of in vitro models that accurately mimic in vivo form and function are necessary (i) to reduce the use of live animal models and their associated financial and welfare costs, (ii) to study complex cellular processes that are difficult to isolate and study in vivo, and (iii) to avoid ethical barriers . Several tissue and cell culture models have been described to study reproductive physiology and pathology, including the study of complex intercellular communication between cells and tissues, involving extracellular vesicles (EVs).Cells in vivo and in cell culture systems communicate extensively using EVs, which are discrete, lipid-bound nanoparticles that carry biological materials, including proteins and nucleic acids. Organoids are an ideal model system to evaluate the EV role in an isolated population of cells because organoids can proliferate long term, while maintaining in vivo-like physiological structure and function. Furthermore, organoids facilitate the evaluation of specific cell types without confounding interactions from other cell types and their secreted EVs that occur in vivo. Organoids provide a controlled system that allows investigators to assess changes in EV secretion and composition from cells, in response to various stimuli. Utilizing organoid cell cultures as a model to evaluate EV function in reproduction has been limited, thus far. Therefore, the goal of this paper is to review and compare organoids to other current in vitro cell culture models used to study female reproductive tissues, with an emphasis on EVs secreted or accepted by reproductive tissue-derived organoids. A literature search was performed between May and July 2021, using NCBI PubMed, including the search terms female and reproduction, and spheroid, organ-on-a-chip, and organoids or extracellular vesicles. Subsequent full text available articles were selected that covered studies that focused only on mammalian female reproduction. Finally, we will explore future directions for organoids in the rapidly developing field of EVs in female reproduction.A variety of in vitro model systems, including monolayer, explant, spheroid, organ-on-a-chip, and organoid  cell culHistorically, the most widely used system to study physiology and/or disease states of female reproduction in vitro were two-dimensional 2D) monolayer culture systems A. MonolaD monolaySeveral 3D cell culture systems have been described . Explant® extracellular matrix [Spheroids are 3D cell clusters that do not require an extracellular matrix to form and cannot self-assemble or proliferate long term D 6]. Si. Si6]. Sr matrix ,11. Under matrix ,11. WhilAnother reproductive tissue that has been challenging to recapitulate with normal physiology in vitro is the placenta. Equine chorionic girdle cells, which are horse-specific specialized trophoblast cells, were cultured in a dish, either with or without 10% fetal calf serum. The chorionic girdle cells cultured with serum adhered to the culture flask as a monolayer, but the cells cultured without serum formed non-adherent spherical clumps, referred to as ‘vesicles’ by the authors . These ‘Another type of 3D cell culture system is one that uses microfluidics and is often referred to as ‘organs-on-a-chip’ . TheOrganoids are grown from stem cells or organ progenitor cells, which are a more differentiated type of stem cell, that self-assemble and remain both genetically and phenotypically stable throughout long-term (months) culture ,23 FiguE. The gePAX2, a gene associated with Müllerian duct cells of secretory function, is expressed in cells toward the uterotubal junction but not toward the infundibulum, and the WT1 gene and protein expression, which is associated with normal cell development and survival, is expressed toward the infundibulum but not toward the uterotubal junction [There are a limited number of reports on female reproductive tissue-derived organoids , and onljunction . These fjunction ,30. Thisjunction .BRCA1 mutation, that maintained cellular abnormalities associated with serous tubal intraepithelial carcinoma for at least four months in culture [Oviductal organoids also have been reported to mimic pathological conditions. One group utilized organoids developed from human-induced pluripotent stem cell (iPSC) lines, with  culture . Further culture , which sPTGS2, PGES, and OXTR, which are genes associated with the prostaglandin synthesis cascade [Organoids derived from endometrial epithelial cells have been described in mice, women, domestic horses, and endangered equids ,34,35,36 cascade .®. Culture of endometrial epithelial cell organoids on a scaffold with stromal cell seeding was reported only up to 10 days [Limitations of these endometrial organoid models are the lack of stromal cells and the extracellular matrix requirement, which is often of animal cell origin and has batch variability. As stromal and epithelial cells comprise the primary cell types associated with the endometrium, in vitro cell cultures are more similar to in vivo if they reflect this multi-cellular environment. To address these limitations, organoid co-culture of endometrial epithelial and stromal cells using 3D porous collagen scaffolds  or agaro 10 days . With th 10 days . Cellula+) also was elevated in the intra-organoid fluid compared to the extra-organoid fluid, which was demonstrated to be necessary in mice during pregnancy, to prevent congenital malformations and miscarriages [Finally, human endometrial epithelial organoids were utilized to demonstrate that fluid produced by the apical side of the organoids into the organoid lumen is biochemically different from fluid produced by the basal side of the organoid into the surrounding conditioned media, with 17 unique metabolites in the intra-organoid fluid and 69 unique metabolites in the extra-organoid fluid , which iarriages ,41. Initarriages .HOX genes and their cofactors, and found that organoids developed with endometriosis tissues maintained the epigenetic patterns for the majority of the evaluated sites [Several studies have utilized organoids derived from human endometrium as model systems for pathologies and to evaluate endometrial receptivity. One study developed organoids utilizing endometrium collected from healthy women and those with adenomyosis to study infertility and determined that organoids generated using the pathological tissues maintained their diseased phenotype . Anothered sites . Thus, eed sites . A furthed sites . This gred sites . The samed sites . This daed sites . All of ® and overlaid with culture media, containing various supplements [Placental cell  organoids have been reported as a model to study human placentation ,48. Firsplements ,48. Orgaplements ,49,50. TSeveral groups have recently reported the culture of organoids derived from various ovarian neoplasia subtypes ,52,53,54Organoids provide a physiologically-representative, less labor-intensive in vitro model, for the study of reproductive physiology, pathology, and therapeutics. Future studies utilizing a 3D organoid system may involve the improvement of in vitro maturation of oocytes in many species, using co-culture with oviductal organoids, or in vitro culture of embryos, utilizing co-culture with oviductal and/or endometrial organoids. This may be particularly applicable for the currently unconquered canine in vitro maturation system  or unaccThis in vitro culture model also can be used effectively to study complex intercellular communication interactions and pathways, particularly the role of EVs in normal and pathological reproductive organ systems. Having a functional, long-term in vitro model that better recapitulates in vivo conditions than traditional in vitro models offers significant advantages when studying normal and pathological female reproductive processes by (i) reducing time requirements by performing evaluations concurrently rather than consecutively, (ii) lessening financial cost due to the high expenses of utilizing research animals, and (iii) improving research animal welfare.Intercellular communication in reproductive tissues involves many processes, including direct cellular interaction through gap junctions, autocrine and paracrine signaling, endocrine communication with hormones, and communication through EVs. The standard guidelines for EV terminology, physiology, separation, and characterization are an important component for understanding the biogenesis of organoid-derived EVs and their potential research and therapeutic roles. EVs are nanoparticles that contain bioactive molecules, such as nucleic acids and proteins, and are surrounded by a lipid bilayer. They cannot replicate  and are naturally secreted by cells, and their primary function is intercellular communication . HistoriEVs play a pivotal role in female reproduction  and areUtilizing in vitro models, the role of EVs from specific cells and/or tissue types can be evaluated more effectively than in vivo and in a more sustainable manner. EVs secreted by bovine amniotic cell monolayer cultures improved the hatching percentage and pregnancy rates of cryopreserved in vitro-produced embryos, compared to cryopreserved embryos not treated with amniotic EVs, by modulating the expression of specific miRNAs . The samCo-culture of EVs secreted by estrual oviductal spheroids with canine oocytes improved in vitro maturation to metaphase II , which oWhile there are many reports on the study of EVs in vitro, there are few reports on the presence, role, or function of EVs derived from cells in organoid models, especially organoids derived from reproductive tissues. In vitro models are necessary for the evaluation of EV content and/or function because the role of EVs, produced by specific cells or tissue types under specific conditions, cannot be studied in vivo, effectively, without confounding factors. For example, EVs secreted by mural granulosa cells in ovarian follicles can be best studied in vitro, without EV contamination from cumulus cells.Most research evaluating EVs secreted by organoids has focused on cancer, cardiac repair, and stem cells , but theSeveral groups have evaluated EVs secreted by neoplastic cells to understand the pathophysiology of neoplastic conditions to develop novel diagnostic tools. Co-incubation of EVs secreted by esophageal adenocarcinoma with a gastric organoid model resulted in the induction of a neoplastic phenotype, which revealed specific neoplastic-inducing miRNAs in the EV cargo , which wTrichuris muris) to evaluate host–parasite interactions [Intestinal organoids are another field that has demonstrated the wide uses for this model system. Intestinal organoids have been used to demonstrate that EVs play a role in inflammatory immune modulation and that opioids impair immune modulation by the secreted EVs . Anotherractions . These f® technology. While these preliminary data demonstrate that reproductive tissue organoids secrete EVs, a large gap in our knowledge exists regarding the cargo and function of these EVs and the utility to study these in organoid systems of female reproductive physiology.Evaluating EVs from a specific cell type is not possible in vivo, but a relevant, physiological, in vitro 3D model can facilitate this. EVs derived from bovine oviductal fluid (in vivo) versus bovine oviductal epithelial cells, cultured in a 2D monolayer (in vitro), revealed differences between the protein content of these two EV sources , which iTissue interactions may be more effectively evaluated in vitro than in vivo utilizing EVs. Placental EVs could be added to endometrial organoid cell cultures to determine the signaling role between the conceptus and the uterine environment. Furthermore, placental EVs in women can be found in the maternal plasma , which iAnother area to explore is the expected EV production per organoid. The EVs produced per organoid are likely dependent on the cell origin and total cells per organoid, which might be determined by extrapolating from the organoid diameter. This is important because EV production per organoid could have significant implications for the commercial production of EVs for therapeutics.Production and separation of EVs secreted by organoids can be time-intensive to generate a large number of EVs, which would be required for commercial production for therapeutic use. Alternative models for high-throughput production of EVs are needed. Bioreactors may be a culture method that can increase the total number of organoids and EVs produced and reduce labor input, to facilitate the commercial application of organoid/EV biotechnology. Bioreactors have been demonstrated to improve the functionality of organoids , and anoAdditional automation technologies could also increase organoid throughput. Several commercial companies have reported 3D bioprinters for automating the extracellular matrix plating process, which traditionally relies on the speed of the technician to maintain temperature control of the extracellular matrix, and accuracy of the technician to distribute extracellular matrix droplets in the appropriate orientation in the culture dish. Automation increases the speed, accuracy and, ultimately, performance of these extracellular matrices to increase organoid and EV production.As in vivo-produced EVs from the reproductive tract improve various sperm parameters, oocyte maturation, and pregnancy rates after the transfer of EV-treated embryos, an alternative in vitro source of EVs from a physiologically relevant model, such as organoids, would facilitate the sustainable and commercial use of EVs for artificial reproductive technologies. Production of EVs in vitro can be scaled to meet clinical in vivo needs, while only a finite amount of EVs can be collected from in vivo sources. Additional research will be required to determine whether large-scale, commercial production of EVs is ideal or if individualized EV production is a more appropriate objective. One concern that must be evaluated prior to the use of in vitro-produced EVs as an in vivo therapeutic is the potential epigenetic alteration that may occur as a result of these EV treatments. A recent publication, using a mouse model with some supporting human data, demonstrated that EVs produced by stressed epididymal cells deliver miRNA to sperm that, ultimately, result in offspring that display a physiological stress phenotype . These lOrganoids also may be utilized to treat reproductive pathologies or to promote the likelihood of pregnancy. Similar to how a tissue’s microbiome must be maintained for healthy organ function, perhaps a healthy tissue secretes a specific population of beneficial EVs. If this is true, perhaps EVs from healthy reproductive tissue, such as from the uterus, may be supplemented to tissues/organs that have undergone pathological changes associated with disease or infection, such as endometritis. Furthermore, EVs may be involved in maternal recognition of pregnancy (MRP) signals ,103. In The role of organoids in personalized medicine is a rapidly emerging area of research. Tissue from a specific patient could be grown in an organoid culture system and various therapeutics could be studied in tandem to determine the most appropriate treatment for that patient’s pathology. This would be similar to bacterial culture from the endometrium of an individual with endometritis, followed by sensitivity trials with a range of antibiotics, to determine the optimum therapeutic. Another case for individualized medicine would be the collection and evaluation of EVs secreted by organoids derived from a pathology to determine appropriate biomarkers in a liquid biopsy for a specific pathology. To accomplish this, a fluid sample, such as blood or urine, would be collected, the EVs in the sample evaluated, and a pathology associated with certain EV cargo or surface molecules may be diagnosed in lieu of an invasive tissue biopsy.The hemochorial placenta in humans is the most invasive type of placentation and allows proteins, such as those associated with immunity, to cross readily from the mother into the fetal blood. Domestic animals have less invasion of the placenta into the maternal endometrial cell layers, such as endotheliochorial, synepitheliochorial, and epitheliochorial placentation, which allows very little protein to cross between the dam and fetus. However, as EVs are lipid-bound nanoparticles, they likely have a greater ability to cross between the fetus and dam in domestic animals. How much access EVs have to cross between the placental layers of the dam and fetus for each non-human placental type has yet to be evaluated. Depending on the amount of EV crossover, EVs in domestic animal maternal blood may be a biomarker for fetal or placental health . These EIn addition, gestational aging may be possible by measurement of blood EV concentration , dependi3D in vitro cell culture models have been utilized for many years and have evolved to more closely mimic in vivo reproductive anatomy and physiology. Recently, new applications of cell culture technologies, such as utilizing extracellular matrices in a 3D droplet, in conjunction with specific culture media to allow organoid generation, have facilitated the applicability of these 3D models to study reproductive physiology and pathophysiology. Development of organoids from reproductive tissues in humans and domestic and wildlife species has been limited, despite the financial, welfare, and time benefits they offer over in vivo models. In particular, 3D cell culture models in wildlife species have greatly improved the safety of both animals and their human caretakers. Utilizing organoids for the study of specific EV functions is promising in the field of reproduction. EVs produced by female reproductive organoids can be used diagnostically, therapeutically, and to improve understanding of normal and pathological physiology of reproduction in many species.


Cancers arising from the lining of the uterus, endometrial cancers, are the most common gynecologic malignancy in the United States. Once endometrial cancer escapes the uterus and grows in distant locations, there are limited therapeutic options. The most aggressive and lethal endometrial cancers carry alterations in the protein p53, which is a critical guardian of many cellular functions. The role of these p53 alterations in endometrial cancer is not well understood. The goal of this work was to use p53 altered models of endometrial cancer to understand which, if any, therapeutically targetable vulnerabilities these p53 alterations may confer in endometrial cancer. Here we show that many of these p53 altered cells have problems with cell division which can be targeted with novel single and combination therapies. These discoveries may lead to relevant new therapies for difficult to treat advanced stage endometrial cancers.TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States.  TP53 mutant, being the most lethal and difficult to treat . Mo. MoTP53 s (PDOs) . TP53 muectively A,B . A. A31]. AOverall, the focused sensitivities to AZD1775 and the common sensitivity to paclitaxel suggested the potential for a cell cycle checkpoint defect in these cells. The focused sensitivities to AZD6738, AZD1775, and gemcitabine also suggested that these tumors may have mechanistically different replication fork protection defects best detected by challenging cells with each agent and then comparing them.TP53 mutant uterine serous carcinomas . In. In32]. Thus, we treated the same cell lines with AZD1775 and harvested for cell cycle flow cytometry analysis at various timepoints A. For eaThe cell lines were again treated with AZD1775 and assessed for histone H3 phosphorylated on serine 10 (H3pS10) to mark mitosis at various timepoints after treatment . Each liThe decreased ability of ARK1 and SPEC2 to arrest in G2/M compared to HEC1B, combined with no clear difference between these three cell lines in their ability to upregulate the replication stress response, hinted that the underlying functional defect in ARK1 and SPEC2 is likely in a part of cell cycle checkpoint regulation not targeted by WEE1. The next question was what the mechanism of the checkpoint defect, if any, was in ARK1 and SPEC2 cells.To determine if other cell cycle pathway defects might influence AZD1775 sensitivity or if there was another molecular defect in these tumors, we studied the transcriptional profiles of cells with varying sensitivity to AZD1775. We profiled a panel of our organoids including resistant (19-99), moderately sensitive (DF-85), and highly sensitive (19-105) lines. Each organoid line was treated with AZD1775 over a time course. Western blots were performed to assess WEE1 engagement  and replication stress response upregulation  in each line. As with cell lines, each organoid line had successful WEE1 engagement shown by pCDC2 downregulation but not RRM2 alteration, and successful upregulation of the replication stress response shown by upregulation of pRPA, γH2AX, pKAP1, and pCHK1 at varying times after treatment . A vehicAURKA)  overrepresentation analysis on post-AZD1775 treatment upregulated genes revealed marked upregulation in G2/M related pathways  25], mo, mo25], AURKA) . Given tAURKA) A and theAURKA) , we hypoTo test this, we treated our models with Alisertib MLN8237), which inhibits Aurora kinase A and to a lesser extent Aurora kinase B at different concentrations  34,35].,35.34,35, which iSince Alisertib targets both Aurora A and Aurora B kinases at different doses, we tested a subset of the cell lines for cell cycle arresting capacity and sensitivity to the Aurora A specific inhibitor MK5108 and the Aurora B specific inhibitor Barasertib (AZD1152) to determine if inhibition of one or both kinases was more effective  34,35].,35.34,35TP53 mutations in our UC cells (CDKN1A) after AZD1775 treatment  in HEC1Bs, which should bind and block the function of any p53, to determine whether losing p53 function altered Alisertib sensitivity H 47]. T. T47]. TTP53 mutant UCs we treated ARK1 with Alisertib as HEC1B does not express p21 ,2320,23]To address this, we treated ARK1 and HEC1B with either a dose curve of Alisertib, the pan-AKT inhibitor Ipatasertib, or Alisertib combined with Ipatasertib, hypothesizing that if AKT plays an active role in the cellular response to Alisertib, then its inhibition should alter Alisertib sensitivity B 48,49],4948,49]Taken together, these data suggested that (1) both Alisertib sensitive (ARK1 and SPEC2) and resistant (HEC1B) cells rely upon LKB1–p53–AKT interconnected signaling, and (2) Alisertib engages the p53 and AKT arms of the LKB1 pathway simultaneously likely utilizing the p53 arm to induce cell death and blocking the protective AKT arm. However, Alisertib sensitivity requires an additional functional defect as evidenced by the resistance of HEC1B when compared to ARK1 or SPEC2. Consequently, our next question was what the additional defect mediating Alisertib sensitivity was and how Alisertib resistance in cells lacking such a defect, like HEC1B, can be overcome.TP53 mutation status, combining WEE1 and Aurora kinase inhibitors leads to enhanced cell death over either agent alone because WEE1 inhibition triggers mitotic progression in cells with Alisertib induced spindle defects [TP53 mutant UC cells. In those with intact G2/M checkpoints, like HEC1B, WEE1 inhibitor-induced mitotic progression is needed in tandem with Aurora kinase inhibitor-induced spindle defects for more efficient cell death.HEC1B demonstrated the strongest cell cycle checkpoint arrest in response to WEE1 or Aurora kinase inhibition , and we  defects . We specTo test this, we treated HEC1B, ARK1, SPEC2, and AN3CA with either Alisertib, AZD1775, or Alisertib+AZD1775 and assessed cell survival C. In theThis increased sensitivity in the resistant HEC1B line and to a smaller extent in the three Alisertib sensitive lines could be due to (1) the combination of DNA damage induced by AZD1775 with mitotic irregularities induced by Alisertib, or (2) the combination of progression into mitosis induced by AZD1775 with mitotic irregularities induced by Alisertib. To determine which occurred, all four lines were treated with vehicle, Alisertib, AZD1775, or Alisertib+AZD1775 and assessed for replication stress response and cell cycle proteins by western blot . All linTo test this, AN3CA, ARK1, SPEC2, and HEC1B were treated with vehicle, Alisertib, AZD1775, or Alisertib+AZD1775, and cell cycle progression was assessed by flow cytometry analysis. HEC1B, ARK1, and AN3CA all showed significant G2/M arrest with both Alisertib or Alisertib combined with AZD1775 while SPEC2 did not, reflecting its inability to arrest after any challenge to G2 or M progression . For HECTo better assess mitotic figure formation, mitotic catastrophe, or apoptotic blebbing post-treatment, AN3CA, ARK1, SPEC2, and HEC1B were treated with vehicle, Alisertib, AZD1775, or Alisertib+AZD1775 and stained for H3pS10 to mark mitosis and tubulin to mark mitotic spindles 24 h after treatment. Cells with normal or Alisertib altered mitotic figures, mitotic catastrophes, and apoptotic blebs were counted in each setting B–D. AlisIn the vehicle treated group, the percentage of mitotic cells was uniform near 5% for all four cell lines C. For AZFor Alisertib, we expect to see significantly more mitotic figures and potentially some level of apoptosis based on flow cytometry and western analysis A. All foFinally, for the Alisertib+AZD1775 combination, we saw some percentage of apoptosis, mitotic figures, and catastrophes in all four cell lines C. KnowinTo demonstrate the increased apoptosis induced by the combination in HEC1B cells beyond just what was visible by eye, HEC1B were treated with vehicle, Alisertib, AZD1775, or Alisertib+AZD1775 and assessed for increased apoptosis by flow cytometry and cleaved PARP western blot 24 h after treatment. The combination was the only treatment to induce significant apoptosis over control by flow cytometry in HEC1B E, and atTP53 mutations in UC has not been well defined. Here, using a panel of TP53 mutant UC cell lines and PDOs, we determined the key functional defect in these tumors is not in the replication stress DNA damage response but rather in mitosis and G2/M cell cycle checkpoints. This defect reveals a unique dependency in many of these tumors on Aurora kinase–LKB1–p53–AKT mediated cell cycle checkpoint control, often altered in UCs through TP53, PIK3CA, or AKT1 mutations [TP53 mutant UC cell survival and two new therapies to target this mechanism, including Aurora kinase inhibitors either alone or in combination with WEE1 inhibitors.The functional and therapeutic significance of utations ,42. ThisTP53 mutant models. Our data indicate that at baseline many TP53 mutant UC cells, represented here by ARK1, AN3CA, and SPEC2, harbor difficulties with G2/M checkpoints and mitosis yet survive due to a combination of mutant p53 protein function and upregulation of Aurora kinase–LKB1–p53–AKT signaling  with all types of TP53 mutations  to validate this possible biomarker. For those TP53 mutant UCs with intact G2/M checkpoints, combining WEE1 and Aurora kinase inhibitors is an effective strategy. These therapies represent relevant mechanism driven strategies for patients with these difficult to treat tumors who have limited options beyond standard chemotherapy and surgery.Overall, these results reveal many