Photodynamic therapy (PDT) is an anticancer therapeutic modality with remarkable advantages over more conventional approaches. However, PDT is greatly limited by its dependence on external light sources. Given this, PDT would benefit from new systems capable of a light-free and intracellular photodynamic effect. Herein, we evaluated the heavy-atom effect as a strategy to provide anticancer activity to derivatives of coelenterazine, a chemiluminescent single-molecule widespread in marine organisms. Our results indicate that the use of the heavy-atom effect allows these molecules to generate readily available triplet states in a chemiluminescent reaction triggered by a cancer marker. Cytotoxicity assays in different cancer cell lines showed a heavy-atom-dependent anticancer activity, which increased in the substituent order of hydroxyl < chlorine < bromine. Furthermore, it was found that the magnitude of this anticancer activity is also dependent on the tumor type, being more relevant toward breast and prostate cancer. The compounds also showed moderate activity toward neuroblastoma, while showing limited activity toward colon cancer. In conclusion, the present results indicate that the application of the heavy-atom effect to marine coelenterazine could be a promising approach for the future development of new and optimized self-activating and tumor-selective sensitizers for light-free PDT. S0) to its lowest singlet excited state (S1) and will undergo intersystem crossing (ISC) to triplet states (T1), now capable of sensitizing the highly cytotoxic singlet oxygen after reacting with triplet oxygen . ,7. 6,7].1 states . Howeverd states ,11.S0 → T1 ISC pathway can then enable the use of the chemiluminescent reaction of Clz as a self-excitation mechanism to directly generate triplet states able to sensitize singlet oxygen, thus leading to a light-free intracellular reaction exclusively triggered by a cancer marker (superoxide anion). One of the most effective strategies to enhance the efficiency of ISC pathways is through the heavy-atom effect  [a] pyrazin-3(7H)-one (Br-Cla)  . Accordi(Br-Cla) a, a bromectively . Br-Cla ectively ,13. GiveThus, studies with Br-Cla and the remaining brominated Clz derivatives  providedTo further understand the true potential of these Clz derivatives as novel anticancer compounds, it is also important to know how they behave toward different cancer types and assess their spectrum of application. So far, our knowledge in this regard is limited to their application to prostate and breast cancer ,13.S0 → T1 pathway present in the chemiluminescent reaction of these compounds and, hence, favor T1 chemiexcitation in the increasing order of OH < Cl < Br. If the reported anticancer activity of the compounds is indeed related to the intracellular superoxide-triggered T1 chemiexcitation, this should lead to heavy-atom-dependent enhancement of their cytotoxicity in the increasing order of OH < Cl < Br. Thus, these three derivatives were subjected to a detailed and in-depth luminometry, photophysical, and theoretical characterization of their chemiluminescent reactions, focused on the possible superoxide-induced generation of triplet excited states. Subsequently, their cytotoxicity was also evaluated for the first time toward neuroblastoma and colon cancer cell lines, with the resulting performance compared with that obtained for breast and prostate cancer. With this approach, we will be able to understand if the heavy-atom effect is indeed responsible for the anticancer activity of these molecules, supporting their identification as prototypical light-free and self-activating photosensitizers, and to understand the scope of their cytotoxicity toward different cancer types.Herein, we aimed to clarify these topics by employing a target-oriented approach. Specifically, we report the synthesis of three Cla derivatives  basis set. The T1 state was calculated by performing single-point calculations at the same level of theory, on top of the S0 IRC-obtained structures. ωB97XD is a long-range-corrected hybrid exchange-correlation functional, which provides quite good estimates for π → π * and n → π * local excitation, as well as charge transfer and Rydberg states [The nctional  and the g states . Geometrg states . This apg states .1H-NMR and high-resolution mass spectra (HR-MS) for OH-/Cl-Cla are also available in the Br-Cla was prepared following the procedure described in , while ON,N-dimethylformamide (DMF)-acetate buffer pH 5.14 (0.68%) or methanol in the presence of a superoxide anion source . The steady-state chemiluminescent and fluorescent spectra of the three Cla derivatives were measured using a Horiba Jovin Fluoromax 4 spectrofluorimeter, with an integration time of 0.1 s. Slit widths of 5 nm were used for both the excitation and emission monochromators when obtaining fluorescent spectra. Chemiluminescent spectra were obtained with a slit of 29 nm for the emission monochromators. Quartz cells with a 10 mm path length were used.Chemiluminescence kinetic measurements were performed in a homemade luminometer using a Hamamatsu HC135-01 photomultiplier tube. All reactions took place at room temperature at least in sextuplicate. The light-emitting reactions took place at room temperature at least in sextuplicate and were carried out in either 2)) The chemiluminescent and fluorescent spectra were obtained in 2 mL DMF-acetate buffer pH 5.14 (0.68%) solutions and measured using a Horiba Jovin Fluoromax 4 spectrofluorimeter (The detailed procedure is available in the The light-emitting reactions took place at ambient temperature at least in sextuplicate and were carried out in DMF-acetate buffer pH 5.14 (0.68%) or methanol , supplemented with 100 U/mL penicillin/100 µg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS), incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. For cell culture maintenance, cells were subcultured once a week and the cell medium was renewed every 2 days. Both the human prostate (PC-3) and the breast (MCF-7) cancer cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics (complete medium), and they were incubated in a 5% CO2 incubator at 37 °C.The HT-29 and SH-SY5Y cell lines were obtained from ATCC. The human colon cancer HT-29 cell line was cultured at 37 °C and 5% CO2. Controls were composed of DMSO 0.1% v/v. Cells were exposed to the different treatments for 48h. For assays involving PC-3 and MCF-7 lines, cells were treated with Br-Cla at 0.1, 1, 10, 25, 50, and 75 µM (in methanol) for 72 h [v/v. The half-maximal inhibitory concentration (IC50) value was determined for the different assays by MTT viability assay.Before each treatment, HT-29, SH-SY5Y, PC-3, and MCF-7 cells were seeded in 96-well plates in triplicate with the following densities: 5000 cells/well for PC-3 and MCF-7, 15,000 cells/well for HT-29, and 20,000 cells/well for SH-SY5Y. For assays involving HT-29 and SH-SY5Y lines, cells were treated with OH-, Cl-, and Br-Cla (in DMSO) at 0.01, 0.1, 1, 10, 20, 30, 50, 75, or 100 µM for 48 h. During the experimental period, cells were maintained at 37 °C with 5% COfor 72 h . The conAfter cell treatment with the Cla derivatives, mitochondrial function was evaluated since mitochondrial dehydrogenases of living cells can reduce the MTT (yellow) to formazans, which are purple compounds. At the end of incubation, the cell medium was removed and 100 µL of MTT solution (0.5 mg/mL in PBS) was added to each well. Cells were then incubated for 3 h, protected from light. After this period, the MTT solution was removed, and DMSO (100 µL/well) was added to solubilize the formazan crystals. Absorbance was measured at 570 nm using an automated microplate reader . All conditions were performed in triplicate.p-value < 0.05.GraphPad Prism 8  was used to produce concentration-response curves by nonlinear regression analysis. MTT results are presented as the mean ± SEM for n experiments performed. All data were assessed in three independent experiences. Statistical comparisons between control and treatment groups were performed with one-way ANOVA test. Statistical significance was accepted at a Cell morphology was assessed on a Leica DMI 6000B microscope equipped with a Leica DFC350 FX camera and then analyzed with the Leica LAS X imaging software v3.7.4 .T1 chemiexcitation during their chemiluminescent reaction, which can be enhanced by the heavy-atom effect, before their synthesis and characterization. Thus, we calculated the potential energy curves for both the S0 and T1 states during the thermolysis of OH-Cla  [S0 and T1 states during the thermolysis reaction , when reaching the TS and onward, both states become degenerated in a large and flat region of the potential energy surface (PES).As consistent with previous reports for different dioxetanones (including Br-Cla) ,11,12,17reaction  is more T1 chemiexcitation, in line with previous studies for this type of system [S0 → T1 chemiexcitation pathway for OH-Cla is not expected to be efficient [This indicates that both chemiluminescent reactions possess an intrinsic pathway for f system ,11,12,18fficient . Given tT1 state with an energy higher than 0.98 eV (the required energy to convert molecular oxygen into singlet oxygen) [S0 – T1 energy gap of OH-Cla coelenteramide and found a value of 2.78 eV. Furthermore, this value is identical to what was found by us previously for Br-Cla [S0 – T1 energy gap of Br-Cla coelenteramide [It should be noted that an efficient ISC is not the only parameter determining the performance of a photosensitizer in singlet oxygen generation, as it should also have a  oxygen) . We moder Br-Cla . Thus, bteramide .Given that the in silico modeling results indicate that the chemiluminescent reactions of these Cla derivatives present intrinsic pathways for light-free generation of triplet states capable of sensitizing singlet oxygen, we then proceeded to the synthesis of OH-Cla, Cl-Cla, and Br-Cla . The int1H-NMR spectroscopy  with addition of buffer , conditions in which Clz and derivates are known to readily generate chemiluminescence by reacting with dissolved oxygen . All comS0 reaction. However, in our previous study of Br-Cla [−1). Furthermore, the S0 energetics for the thermolysis of OH-Cla and Br-Cla dioxetanones are identical . To verify this hypothesis, we also measured the fluorescence intensity of the spent reaction mixtures (after 30 min of reaction) in an aprotic solvent  in methanol. Interestingly, all compounds responded to the addition of KO2 with immediate emission of light and subsequent decay to basal levels , without affecting the chemiluminescent reaction. Thus, if the anticancer activity found before for this type of molecules ,13 is inGiven this, the potential anticancer activity of the three R-Cla compounds was evaluated in both colon cancer (HT-29) and neuroblastoma (SH-SY5Y) cell lines with a standard MTT assay for exposure times of 48h, along with changes in cell morphology analyzed by microscopy.T1 chemiexcitation. Interestingly, while Cl-Cla induced virtually no anticancer effect in these cells, there was a statistically significant difference at a concentration of 100 µM , in the same concentration range as R-Cla . This drIn neuroblastoma cells , the resT1 chemiexcitation and the subsequent intracellular generation of triplet states capable of sensitizing singlet oxygen [Given this, it is clear that there was a halogen-dependent effect on the anticancer activity of these compounds for both cancer cell lines. This is in line with our proposition that the addition of halogens to Clzs will provide them with anticancer activity, by enhancing t oxygen ,13. In ct oxygen ,13 is inHaving said that, it is also important to note that the intrinsic anticancer activity of the studied compounds (including Br-Cla) might not be as high as desired toward neuroblastoma and colon cancer cell lines, especially when compared to 5-FU. Given this, it is important to determine whether the magnitude of the obtained anticancer activity is similar across different cancer types or if it is dependent on the studied cell line.50 of Br-Cla for different cell lines. Br-Cla was chosen for its consistent toxicity in this study, contrary to OH-Cla and Cl-Cla. IC50 values were determined for neuroblastoma SH-SY5Y, prostate PC-3, and breast MCF-7 cancer cell lines  was still significantly higher than the IC50 found for MCF-7, irrespective of the duration of treatment with Br-Cla.The impact of treatment with Br-Cla on the cellular viability of PC-3 and MCF-7 cell lines can be found in 0 values . More spreatment . This vaThus, the results indicate that it is safe to state that the magnitude of the anticancer activity of Br-Cla is dependent on the cancer cell type, being higher for prostate and breast cancer than for neuroblastoma and colon cancer. Further studies are required to assess if these differences arise from (among other possibilities) higher resistance of HT-29 and SH-SY5Y cell lines to the photodynamic effect, lower efficiency of internalization of R-Cla compounds into these cell lines, or a lower generation of superoxide anion in these cells.S0 → T1 chemiexcitation pathways. The anticancer activity of these compounds was evaluated toward different cancer cell lines, and a clear halogen effect was observed with Cl-Cla and Br-Cla presenting increasing toxicities. Furthermore, it was found that the magnitude of the anticancer activity of these types of compounds is dependent on the cancer cell line, being more relevant in prostate and breast cancer than in neuroblastoma and colon cancer. Thus, the results indicate that applying the heavy-atom effect to marine Clz would be a promising strategy for designing self-activating and light-free photosensitizers. With this design strategy, the Clz system appears to be a prototypical system for optimized photosensitizers to be used in PDT strategies not limited by the need for external light sources.In conclusion, we reported the target-oriented synthesis of three Clz derivatives , to provide anticancer activity to this class of compounds through the heavy-atom effect. On the basis of this strategy, we developed novel compounds able to directly generate readily available triplet excited states with enough energy to sensitize singlet oxygen, in a chemiluminescent reaction triggered by a cancer marker. This was achieved by the introduction of the heavy-atom effect into this system, which enhanced the ISC rate of available Patent PCT/IB2019/053642 (pending)—chemiluminescent imidazopyrazinone-based photosensitizers with available singlet and triplet excited states.


N-acetylglucosaminyltransferase 2, a polylactosamine synthase, is an important member of the glycosyltransferase family. However, the biological function and regulatory mechanism of β3GNT2 in esophageal carcinoma (ESCA) is still poorly understood.Glycosyltransferases play a crucial role in various cancers. β1, 3-The Cancer Genome Atlas and Genotype-Tissue Expression databases were used for gene expression and prognosis analysis. Quantitative real-time PCR, Western blot, and immunohistochemistry were performed to detect the expression of β3GNT2 in ESCA cell lines and tissues. In vitro assays and xenograft tumor models were utilized to evaluate the impact of β3GNT2 on ESCA progression. The downstream effectors and upstream regulators of β3GNT2 were predicted by online software and verified by functional experiments.We found that β3GNT2 was highly expressed in ESCA tissues and positively correlated with poor prognosis in ESCA patients. β3GNT2 expression was closely associated with the tumor size, TNM stage, and overall survival of ESCA patients. Functionally, β3GNT2 promoted ESCA cell growth, migration, and invasion in vitro, as well as tumorigenesis in vivo. Mechanistically, β3GNT2 knockdown decreased the expression of the polylactosamine on EGFR. Knockdown of β3GNT2 also inhibited the JAK/STAT signaling pathway. Meanwhile, the JAK/STAT inhibitor could partly reverse the biological effects caused by β3GNT2 overexpression. Moreover, β3GNT2 expression was positively regulated by CREB1 and negatively regulated by miR-133b. Both CREB1 and miR-133b was involved in the β3GNT2-mediated ESCA progression.Our study, for the first time, reveals the importance of β3GNT2 in ESCA progression and offers a potential therapeutic target for ESCA.The online version contains supplementary material available at 10.1186/s11658-022-00306-y. Esophageal carcinoma (ESCA) is one of the most common cancers around the world . China iGlycosylation is a ubiquitous post-translational modification of proteins , 5. AlteN-acetylglucosaminyltransferase 2 (β3GNT2) was overexpressed in ESCA tissues and correlated with the poor prognosis of ESCA patients. We further investigated the biological function and regulatory mechanism of β3GNT2 in ESCA progression. Our results suggest that β3GNT2 may serve as a potential therapeutic target for ESCA.In this study, The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to identify differentially expressed glycosyltransferases between ESCA tissues and normal tissues. We found that β1, 3-https://tcga-data.nci.nih.gov/tcga/) and GTEx  platforms. Differentially expressed glycosyltransferases, transcription factors, miRNAs were screened using the DESeq2 R package. An adjusted P-value < 0.05 and |log2 fold-change (FC)|> 0.5 were set as the thresholds. Volcano plots and heatmaps were generated using the R package ggplot and pheatmap, respectively. A prognostic nomogram was constructed by R software with the rms package. The Receiver Operating Characteristic (ROC) curves were plotted and the area under the ROC curve was calculated using the R package pROC. Survival prediction was performed using the GEPIA database (http://gepia.cancer-pku.cn/). Gene Set Enrichment Analysis (GSEA) analysis was carried out using the GSEA software (http://www.broadinstitute.org/gsea). Co-expression analysis was determined by using the LinkedOmics platform (http://www.linkedomics.org/).RNA-seq data, miRNA-seq data, and the corresponding clinical information were obtained from the TCGA  between 2008 and 2012. The patients, including 35 males and 30 females, were aged 35–75 years with an average age of 54.62 ± 9.14. None of the patients underwent any other treatment before surgery. Samples were stored at − 80 °C until further processing. The studies involving human participants were approved by the Ethics Committee of the Hubei University of Medicine. All patients provided informed written consent.2. The pcDNA3.1/β3GNT2 plasmid, pcDNA3.1/CREB1 plasmid, empty pcDNA3.1 plasmid, a lentiviral vector expressing β3GNT2 shRNA, a lentiviral vector expressing CREB1 shRNA, control lentiviral vector, miR-133b inhibitor, inhibitor control, miR-133b mimics, and mimics control were constructed by GenePharma . All transfections were conducted with Lipofectamine 3000 reagent  according to the manufacturer’s protocol. Stable clones were selected by puromycin or G418.Human ESCA cell lines TE-1, KYSE150, and KYSE410 were purchased from Procell . These cells were cultured in RPMI-1640 medium  containing 10% FBS at 37 °C, 5% CO−ΔΔCt method. All primers were synthesized by GenePharma. Total protein was isolated using RIPA lysis buffer . Western blot was conducted as previously described [Total RNA was extracted using RNAiso Plus . For mRNA detection, cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara) and qPCR was carried out employing the SYBR-Green PCR master mix (Takara). For miRNA analysis, the One-Step PrimeScript miRNA cDNA synthesis kit (Takara) and the SYBR PrimeScript miRNA RT-PCR Kit (Takara) were used. Data were normalized to GAPDH or U6 based on the 2escribed . PrimaryTissues were fixed in formalin and embedded in paraffin. Afterward, 5 µm sections were prepared for IHC staining. PV-9000 two-step immunohistochemical kit  was used to detect the expression of β3GNT2 protein. The positive signal was visualized by DAB detection Kit (ZSGB-BIO). Slides were read by two pathologists using a Nikon Eclipse E600 microscope . IHC score = intensity score × percentage score . β3GNT2 3 per well) were seeded into a 96-well plate. Absorbance was measured at 450 nm. For colony formation assay, cells were added into a 6-well plate at a density of 5 × 103 cells/well. After 2 weeks, colonies (> 50 cells) were stained with 0.5% crystal violet (Beyotime).Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay . Cells (1 × 104 in 200 μL serum-free medium) were seeded into the upper chamber of the Transwell apparatus . Transwell chambers precoated with or without Matrigel  were used to assess cell invasion and migration, respectively. The lower chamber was filled with 600μL of complete culture medium. After incubation for 36 h, migrated or invaded cells were stained with 0.5% crystal violet.Cells . The percentage of apoptotic cells was defined as the sum of early apoptotic cells and late apoptotic cells. All experimental steps were conducted according to kit manual instructions.Lycopersicon esculentum lectin (LEL) agarose beads  were used to analyze polylactosamine chains on glycoproteins. Cell lysates (0.5 mg) were incubated with 30 μL of LEL-conjugated beads to capture the lectin-glycoprotein complexes. Precipitated glycoproteins were eluted and subjected to Western blot analysis with an anti-EGFR antibody. The EGFR in total lysates was used as the internal loading control.The wild-type or mutant β3GNT2 3′UTR sequences containing the binding sites for miR-133b were cloned into the pmirGLO vector. Besides, the fragments of β3GNT2 promoter with wild-type or mutant CERB1 binding sites were inserted into a pGL3-basic vector. All the above plasmids were constructed by GenePharma. Cells were harvested 48 h after transfection. Luciferase activities were determined using the dual-luciferase reporter assay system .The EZ-Magna ChIP™ Kit  was used for ChIP assay . Detecti7) were subcutaneously injected into nude mice. Tumor volume (0.5 × length × width2) was measured every week. Four weeks after injection, all mice were sacrificed.Female BALB/c nude mice, 4–5 weeks old, were provided by the Animal Center of Hubei University of Medicine. Transfected cells  software . Student’s t-test, Chi-square test, and one-way ANOVA were used to determine statistical significance. A correlation was analyzed using the Pearson correlation coefficient test. Overall survival curves were plotted according to the Kaplan–Meier method. A P-value < 0.05 was considered significant.Using the RNA-seq data from the TCGA and GTEx datasets, we screened differentially expressed glycosyltransferases between ESCA tissues 182 cases) and normal esophageal tissues (666 cases)  and the String database (https://www.string-db.org/) to determine the interactive relationship between β3GNT2 and EGFR   was used. Through bioinformatics analysis, one putative CREB1 binding site was identified in the β3GNT2 promoter region  Fig. A. The asml) Fig. B. Notablml) Fig. C. A posiml) Fig. D. Thus, ml) Fig. E, F. To ion Fig. G. The enion Fig. H. Dual-lion Fig. I. To expion Fig. J–M. In sSince miRNAs are important post-transcriptional modulators of gene expression, we then investigated whether β3GNT2 was regulated by specific miRNAs . Three pThe prognosis of patients with ESCA remains unsatisfactory, despite the use of multiple therapeutic approaches. Further exploration of the diagnostic and prognostic biomarkers for ESCA is warranted. Glycosyltransferases have attracted increasing interest owing to their importance in many biological processes and diseases, including cancer. In the current study, we first found that β3GNT2 expression was upregulated in ESCA. β3GNT2 upregulation was closely associated with the poor prognosis of ESCA patients. Subsequent experiments proved that β3GNT2 contributed to ESCA progression by promoting EGFR glycosylation and activating the JAK-STAT pathway. In addition, CREB1 and miR-133b were critical regulators of β3GNT2 Fig. M. Thus, β3GNTs are a family of crucial glycosyltransferases that synthesize a unique glycan structure known as polylactosamine . AbnormaWe explored the underlying mechanism of β3GNT2 in promoting ESCA progression. Bioinformatics analysis showed that β3GNT2 expression was linked to the JAK-STAT pathway. Numerous studies revealed that the JAK-STAT pathway was involved in cell proliferation, differentiation, apoptosis, angiogenesis, and immune regulation , 22. TheTo explore the potential factors leading to β3GNT2 upregulation in ESCA, the transcription factors of β3GNT2 were predicted by bioinformatics methods. CREB1 was proposed as a candidate because it had a strong positive correlation with β3GNT2 expression in ESCA. CREB1 is a transcription factor that influences multiple cellular processes, such as cell survival, differentiation, and proliferation , 29. In There are, however, several limitations to our study. First, the expression of polylactosamine chains in ESCA tissues is not examined. Second, the association between the JAK-STAT pathway and EGFR signaling has not been elucidated. The specific glycosylation sites of EGFR remain unknown. The effect of EGFR glycosylation on ESCA progression has not yet been elucidated. Additionally, the mechanism of synergy between CREB1 and miR-133b is not clearly understood. It will be interesting to address these questions in future research.Our results confirm that β3GNT2 predicts poor prognosis and acts as an oncogene in ESCA. β3GNT2 can enhance EGFR glycosylation and activate the JAK-STAT pathway. β3GNT2 is transcriptionally upregulated by CREB1. Meanwhile, miR-133b directly targets β3GNT2 and negatively regulates its expression. To our knowledge, this is the first study to explore the biological function, molecular mechanism, and regulatory network of β3GNT2 in ESCA. Thus, β3GNT2 may be a promising prognosis biomarker and a potential therapeutic target for ESCA.Additional file 1: Table S1. Screening of dysregulated glycosyltransferases in ESCA based on the TCGA and GTEx databases.Additional file 2: Fig. S1. Analysis of β3GNT2 protein expression in different ESCA cells by Western blot.Additional file 3: Table S2. Differentially expressed genes correlated with β3GNT2 in ESCA based on the LinkedOmics platform.


However, the therapy is mainly limited by severe acute and chronic graft-versus-host disease (GvHD), both being life-threatening complications after allo-HCT. GvHD develops when donor T cells do not only recognize remaining tumor cells as foreign, but also the recipient’s tissue, leading to a severe inflammatory disease. Typical GvHD target organs include the skin, liver and intestinal tract. Currently all approved strategies for GvHD treatment are immunosuppressive therapies, with the first-line therapy being glucocorticoids. However, therapeutic options for glucocorticoid-refractory patients are still limited. Novel therapeutic approaches, which reduce GvHD severity while preserving GvL activity, are urgently needed. Targeting kinase activity with small molecule inhibitors has shown promising results in preclinical animal models and clinical trials. Well-studied kinase targets in GvHD include Rho-associated coiled-coil-containing kinase 2 (ROCK2), spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) to control B- and T-cell activation in acute and chronic GvHD. Janus Kinase 1 (JAK1) and 2 (JAK2) are among the most intensively studied kinases in GvHD due to their importance in cytokine production and inflammatory cell activation and migration. Here, we discuss the role of kinase inhibition as novel treatment strategies for acute and chronic GvHD after allo-HCT.Allogeneic hematopoietic stem cell transplantation  is a potentially curative therapy for patients suffering from hematological malignancies  Pati, cGvHD) –7. GvHD , cGvHD) , 5, 8–10, cGvHD) . Acute a, cGvHD) . Acute G, cGvHD) , 11. Sta, cGvHD) , 14. Man, cGvHD) –17. TCR , cGvHD) –28. ROCK, cGvHD) , 29–32. survival –35. Basesurvival . In genesurvival . Since Gsurvival –38. Manyvia JAK1/2 and signal transducer and activator of transcription (STAT) pathways are crucial for the stimulation of inflammatory cytokine production and the activation of a variety of immune cells during GvHD onset and progression  1/2. Signaling gression .Acute GvHD: The JAK/STAT signaling pathway has high importance in aGvHD onset and progression, as STAT1 and STAT3 are activated early after disease onset. The signaling plays a pivotal role in mediating T-cell activation and changes of the T-cell phenotype  GvHD and patients with severe intestinal or skin GvHD showed impressive response to JAK1/2 inhibition. Treatment with Ruxolitinib was liked to decreased pro-inflammatory serum cytokine levels and lower numbers of activated T-cells. The overall response rate (ORR) was 81.5 % and the overall survival (OS) of steroid-refractory aGvHD patients receiving Ruxolitinib was higher than ever reported for any other pharmacological therapy . This trChronic GvHD: Besides application in aGvHD, Ruxolitinib treatment was also evaluated for the treatment of glucocorticoid-refractory cGvHD  were published recently . The triDonor T-cell activation and inflammatory cytokine secretion is a hallmark of GvHD after allo-HCT. Activation and effector functions of T-cells are tightly connected to the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling cascade, which is crucial for the regulation of T-cell survival, proliferation, cell cycle progression, differentiation and metabolism Figure 3Acute GvHD: In a preclinical study, transplantation of Mtor-deficient donor T-cells reduced aGvHD severity in mice, whereas more detailed analysis with Raptor-deficient allogeneic T-cells revealed that T-cell-mediated pathogenesis is dependent on mTORC1 but not on mTORC2 . The effect of pacritinib/sirolimus was tested in human MLRs and a xenogeneic GvHD model and consistently suppressed allogeneic T-cell proliferation and GvHD severity  , 118. IbBased on very promising preclinical and clinical trial data, ITK and BTK inhibition with ibrutinib could be a very potent therapy in chronic GvHD. However, additional detailed analysis of the underlying mechanism is necessary to improve the therapeutic success. Since clinical trials focus on cGvHD, further analysis and preclinical models are needed to investigate the role of ITK blockade in aGvHD.The non-receptor cytoplasmic spleen tyrosine kinase (Syk) was hypothesized being an important regulator of GvHD as it has functions in transmitting signals from surface receptors, including Toll-like receptors (TLRs) , Fc receAcute GvHD: In a preclinical murine aGvHD model, daily treatment with Fostamatinib led to significantly improved survival, reduced histopathology scores and reduced pro-inflammatory serum cytokine concentrations. Fostamatinib treatment did not interfere with donor lineage engraftment and immune reconstitution. Syk phosphorylation is rapidly increased upon CD3/CD28 stimulation of T-cells and higher pSyk 525/526 levels were also seen in T-cells isolated from aGvHD mice. Using luciferase transgenic T-cells, Syk inhibitor treatment was found to reduce alloreactive donor T-cell expansion in vivo. The findings were confirmed by CFSE staining, indicating reduced T-cell proliferation in vivo upon Syk inhibition. Besides blockade of T-cell proliferation, Syk inhibition also reduced T-cell migration towards CXCL12. Contrary to previous findings about the importance of Syk in T-cell lineage commitment, Fostamatinib treatment did not change the percentage of Th2 and Th17 cells after allo-HCT. Effects on T-cells could be further affected by APCs as Syk inhibition was also connected to diminished costimulatory molecule expression and reduced DC migration in vivo and in vitro. Although proliferation and effector cytokine secretion of allogeneic donor T-cells was significantly reduced by Fostamatinib, the GvL effect was preserved, as confirmed by in vivo bioluminescence imaging using luciferase transgenic leukemia cells and ex vivo killing assays. Overall, pharmacological inhibition of Syk was found being a novel treatment strategy in aGvHD by reducing T-cell expansion and costimulation while preserving anti-leukemia immunity . Another phase II trial was investigating the efficacy and tolerability of entospletinib in combination with systemic corticosteroids cGvHD as first-line therapy, however, the study was terminated (NCT02701634). Based on pre-clinical studies, it would also be interesting to evaluate effects of Syk inhibition in patients with aGvHD. However, ongoing clinical trials first focused on cGvHD, which might be due to the importance of Syk in BCR downstream signaling, whereas it is not essential for TCR downstream events.Taken together, the data derived from genetic and pharmacological approaches in pre-clinical murine GvHD models clearly indicate that Syk plays an important role in GvHD pathophysiology. Pharmacological targeting of Syk could be a novel attempt to treat GvHD. Based on these promising findings of Syk inhibition with Fostamatinib Chronic GvHD: cGvHD is often presented with dermal fibrosis and sclerosis, associated with the presence of stimulatory anti-platelet-derived growth factor receptor (PDGFR) antibodies, suggesting a direct link between skin fibrosis and PDGF signaling  levels. Calcium influx is essential for activation, maturation and effector functions of T-cells. TCR engagement leads to the activation of PLC-γ, thereby increasing the intracellular levels of inositol 1,4,5-triphosphate (IP3). Binding of IP3 to its specific receptors in turn stimulates the release of calcium from intracellular storage compartments. Continuous depletion of intracellular calcium storage stimulates the opening of cell membrane based calcium channels to enhance the influx of calcium from the extracellular compartment. The intracellular increase of Ca2+ is required to activate calcium-dependent kinases and the transcription factor calcineurin, leading to activation of nuclear factor of activated T-cells (NFAT), thereby enhancing the transcription of a variety of different genes necessary for T-cell activation and effector functions . The modulation of intracellular calcium levels was therefore hypothesized to be a potential therapeutic strategy for autoimmune diseases  family, comprising Itpka, Itpkb, Itpkc and inositol polyphosphate multikinase. The Itpk family acts as a negative regulator of Ca2+ influx through conversion of IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4) and this regulatory mechanism is known to be highly important for T-cell development and survival  and interferon γ (IFNγ) levels compared to Itpkb wildtype T-cells. However, quantitative T-cell analysis in the spleen, mesenteric lymph nodes and the small intestine and liver revealed lower numbers of Itpkb deficient T-cells compared to the WT control, whereas the proliferative capacity remained unchanged. Reduced numbers of donor Itpkb-/- T-cells could be linked to lower T-cell survival, indicated by higher abundance of active caspase 8 in CD4+ and CD8+ T-cells from Itpkb-/- donors after HCT , followed by analysis of cytokine production. Pro-inflammatory cytokine secretion was suppressed in a concentration-dependent manner. Application of OZ in a major mismatch murine aGvHD model prolonged the survival of GvHD mice, reduced GvHD scores and did result in lower serum levels of pro-inflammatory cytokines  is a major control mechanism for cellular responses, proliferation and cytokine production , 186. DiChronic GvHD: Since enhanced p38 MAPK phosphorylation was found in fibroblasts from systemic sclerosis patients and p38 MAPK blockade reduced elevated type I collagen expression, the signaling pathway was hypothesized playing a role in sclerosis pathogenesis  in donor grafts were found to accelerate acute intestinal GvHD in mice  – SFB-1479 – Project ID: 441891347, SFB TRR167, SFB850 (to RZ), by the Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984 to RZ) and by ERC Consolidator grant (681012 GvHDCure to RZ). The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding program Open Access Publishing.RZ received honoraria from Novartis, Incyte and Mallinckrodt.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.


Multicellular organisms potentially show a large degree of diversity in reproductive strategies, producing offspring with varying sizes and compositions compared to their unicellular ancestors. In reality, only a few of these reproductive strategies are prevalent. To understand why this could be the case, we develop a stage-structured population model to probe the evolutionary growth advantages of reproductive strategies in incipient multicellular organisms. The performance of reproductive strategies is evaluated by the growth rates of the corresponding populations. We identify the optimal reproductive strategy, leading to the largest growth rate for a population. Considering the effects of organism size and cellular interaction, we found that distinct reproductive strategies could perform uniquely or equally well under different conditions. If a single reproductive strategy is optimal, it is binary splitting, dividing into two parts. Our results show that organism size and cellular interaction can play crucial roles in shaping reproductive strategies in nascent multicellularity. Our model sheds light on understanding the mechanism driving the evolution of reproductive strategies in incipient multicellularity. Beyond multicellularity, our results imply that a crucial factor in the evolution of unicellular species’ reproductive strategies is organism size. With The origin and the evolution of reproductive strategies are not well understood. Only a few reproductive strategies have been considered in previous work. The fragmentation mode of producing many-celled propagules has been investigated, in order to understand cell death in yeast  or to unOrganism size confers various advantages to organisms ,23, suchSaccharomyces cerevisiae [Volvox, along with the germ–soma differentiation [Previous studies have shown that cellular interactions can change reproductive modes ,23,28. Frevisiae . Phenotyrevisiae ,35. In tntiation , 1–20 gentiation .Thus, the size and composition could affect growth in phenotypically heterogeneous multicellular organisms. We develop a theoretical model to address the evolution of reproductive strategies considering the effects of organism size and thresholds for the number of different cell types. The size effects could increase or decrease organism growth, with the organism growing faster when the cell number of a particular phenotype reaches a given threshold. Organisms in a population share one common reproductive strategy while populations differ in reproductive strategies. Reproductive strategies thus compete with each other via population growth rates. The optimal reproductive strategy maximizes the population growth rate. We found that reproductive strategies can coexist or dominate others under different conditions. The uniquely optimal reproductive strategy always produces two offspring units.. 2a). The organisms in each population have a unique reproductive strategy. For example, a population with maturity size N = 3 must have the reproductive strategy either 1 + 1 + 1 or 2 + 1. In a population with 2 + 1, mature organisms with three cells produce a single-celled newborn organism and a two-celled newborn organism. The reproductive strategy determines the organism size at which an organism is born and at which size it is mature and reproduces. For the reproductive strategy n1 + n2 + · · · + nM, newborn organisms have cell number ni  and maturity size n1 ≥ n2 ≥ · · · ≥ nM. We consider organisms consisting of two cell types: type A and type B. Newborn organisms may differ in their size and composition in a population. For example, a population with reproductive strategy 2 + 1 can have five types of newborn organisms: , , ,  and , where  shows the number of type A cells and type B cells in an organism (d). Each organism grows incrementally by one cell at a time. During each increment, a cell is selected to divide, and two daughter cells are produced. Each daughter cell can switch to another phenotype independently with a cell-type switching probability, which is typically m = 0.01 in our model . After reaching their maturity size N, organisms reproduce via random fragmentation in terms of organism composition. The probabilities of forming different newborn organisms are calculated in electronic supplementary material, appendix S1. The newborn organism follows the same life cycle, growing from newborn to mature (see a).We consider multiple populations in which organisms grow and fragment into smaller pieces a. The ororganism d. Each oture see a.Figureλ, depending on the number of offspring and the growth time of organisms [tn is the time for organisms growing from size n to (n + 1). The growth time depends on the cell size at the newborn stage and the cell size at the maturity stage. The time tn is determined by the organism size and composition astsn and tgn are the size component and the threshold component of tn. Next, we discuss how we model tsn and tgn.We assume that organisms in populations grow independently without density dependence. Thus, populations follow exponential growth . The poprganisms ,38, can tsn depends only on the cell number n = nA + nB of an organism during growth, but not on nA or nB individually. Under the neutral condition b). Without loss of generality, we choose γ = 1 and treat tsn around the neutral condition (a). We call n = 1, …, N. By perturbing all normalized cell increment components χn for n = 1, …, 7, we can capture all possible effects of size. For χn = 1, we recover the neutral condition.The size component ism size . Thus, osn0) see a. We caltgn is determined by the number of the cell type of interest. Without loss of generality, we choose the type B. We assume the type B providing a benefit to the organism while bearing an individual cost. An organism grows faster if the number of its type B cells meets a given threshold k (c). There are many methods to construct such a compositional threshold effect. Here, we choose a volunteer dilemma game [n cells with nA type A cells and nB type B cells. When nB meets a contribution threshold k, each cell of the organism gets a benefit b from an increased organism growth. Each B cell pays an individual cost c and A cells pay no costs (d):pA and pB are the division probabilities for A cells and B cells, respectively, and w is the intensity of selection [tgn is the inverse of average fitness of the organism:k.The threshold component eshold k c. There mma game . Consideno costs d:2.2PA. If the organisms of a population are going through a perturbed state at size n, i.e. n1 + n2 + · · · + nM) can deviate from the one under the neutral condition. Since the population growth rate is inversely proportional to growth time, a perturbation is either advantageous  or disadvantageous  for population growth. A reproductive strategy is referred to as being promoted (suppressed) when its population growth rate is greater  than the neutral growth rate 1. A single advantageous perturbation (χn < 1) promotes the reproductive strategy of any population with organisms going through the state n of the perturbation, i.e. the strategies are satisfying nM ≤ n ≤ N (b). The performance of reproductive strategies is unaffected when their populations’ organisms do not go through the size under perturbations, i.e. n < nM or n > N. A single adverse perturbation χn > 1 suppresses reproductive strategies that satisfy nM ≤ n ≤ N. Among these affected populations, we found that the reproductive strategy n + 1 is most affected by perturbations at size n. Since the population with reproductive strategy n + 1 contains n-celled newborn organisms, which mature at size n + 1, its growth time depends on χn. Therefore, under the condition of χn < 1 and χk = 1 , the reproductive strategy n + 1 is uniquely optimal. Under the condition of χn > 1, the reproductive strategy n + 1 is most suppressed (see b). Analogous to the reproductive strategy n + 1, the reproductive strategy n + 2 is the second most affected reproductive strategy. Similarly, for the rest of the reproductive strategies, their population composition determines whether the growth rates are affected or not. The growth rates then determine the performance of reproductive strategies.To focus on size effects only, we first assume no threshold effect,  1, …, 7 a. If the ≤ n ≤ N b. The pessed see b. Analogn = 1, …, 7, which forms a discrete size function with respect to n. As each χn is independently perturbed from the neutral condition, the discrete function can cover all possible shapes. We investigate the general features of size functions that promote a given reproductive strategy, but ignore the specific shape of the size functions. As expected, we observed that the populations of optimal reproductive strategies contain organisms that mostly go through sizes with smaller χn. This is illustrated in figure 2c and an analytical proof is given in electronic supplementary material, appendix S3, for reproductive strategies with N ≤ 3. We found that only the binary-splitting reproductive strategy (producing two offspring) can be uniquely optimal . Intuitively, this is because the fastest-growing newborn organisms in a population with a multiple-splitting reproductive strategy can also be found in another population with a binary-splitting reproductive strategy. For example, the population growth rate of 2 + 1 + 1 cannot be greater than that of 1 + 1 and 2 + 2 at the same time. Additionally, 1 + 1 is the most frequently observed reproductive strategy in binary-splitting reproductive strategies (figure 2d) because 1 + 1 is the only reproductive strategy that depends on a single cell increment component χ1. Therefore, for a randomly chosen χn , 1 + 1 has a higher probability of being optimal compared to other strategies. Generally, reproductive strategies have lower chances to be optimal when binary splitting makes organisms go through many cell increment stages.We can analyse all possible size effects by combining single perturbations for . 3.2χn = 1, such that b − c = 0, cells of type B would never lead to any growth advantage; thus we focus on b − c > 0. With a threshold at size k, newborn organisms of a population with nB ≥ k have larger payoffs and thus have shorter growth time; see equations (d). With the contribution threshold k = 2,  grows fastest as it has two B cells.  is the second-fastest-growing newborn organism as it most likely gains benefits by producing a second B cell during growth. ,  and  grow relatively slowly because they are less likely to produce at least two B cells during growth. For convenience, we refer to newborn organisms in a population as ‘beneficial’ if nB ≥ k and ‘intermediate beneficial’ if nA = 0, nB < k and N > k, where N is maturity size. All other newborn organisms are unlikely to reap the benefits of cooperation before maturity. The growth rate of a population depends primarily on its beneficial newborn organisms and secondly on its intermediate beneficial newborn organisms. Moreover, among all beneficial newborn organisms, the one with exactly k type B cells has the largest payoff. Thus, they have growing advantages under no or low cell-type switching probability. For a low cell-type switching probability, e.g. m = 0.01, homogeneous newborn organisms are more abundant than heterogeneous ones. In the long run, we expect that populations mostly contain homogeneous newborn organisms.To investigate threshold effects exclusively, we assume the size effect to be neutral: quations  and (2.4quations d. With ta). The optimal reproductive strategies can be classified into three categories: multiple optima, symmetric binary splitting (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) and asymmetric binary splitting with a k-celled newborn organism (N − k) + k. When k = 1, multiple reproductive strategies are optimal at the same time (see a–c). Since every population contains beneficial newborn organisms, the performances of different reproductive strategies are similar. As k increases, the symmetric binary-splitting reproductive strategies (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) are optimal for 1 < k ≤ (1/2)N . Newborn organisms with size equal to or greater than k have growth advantages; thus, intuitively (N/2) + (N/2) and k + (k + 1) should have the same performance in population growth. However, we found that only (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) is optimal. The intrinsic composition of the population and the effects of cell-type switching probability m = 0.01 determine the results. To understand the growth advantages of the symmetric binary-splitting reproductive strategies with the maximal maturity size, we take 4 + 4 and 3 + 3 at k = 3 as an example. For k = 3, the population of 4 + 4 contains the beneficial newborn organisms  and . The population of 3 + 3 only contains beneficial newborn organisms . When a cell-type switching event happens during growth,  produces a beneficial newborn organism , while  reproduces a non-beneficial newborn organism . Populations with larger maturity sizes are less affected by the cell-type switching probability as they contain multiple types of beneficial newborn organisms. Finally, when N − k) + k becomes optimal (see a). When N − k) + k rather than other reproductive strategies such as N − k − 1) + k + 1. Because of N − k < k, organisms with N − k cells can only form intermediate beneficial newborn organisms—and only when they are pure B cells. Larger intermediate beneficial newborns grow faster than smaller ones. We take 3 + 1 + 1 and 3 + 2 for k = 3 as an example. 3 + 1 + 1 has the intermediate beneficial newborn organism  and 3 + 2 has the intermediate beneficial newborn organism . During organism growth,  undergoes two cell increment stages with longer time N see a,b. Newbimal see a. When kquations  and 2.2a. The opk, resulting from reducing the number of beneficial and intermediate beneficial newborn organisms. Especially when k ≥ N, no reproductive strategies will obtain the benefits of cooperation, and their populations grow slower due to the associated costs . Increasing maturity size N increases population growth rates of the optimal reproductive strategies because the number of beneficial or intermediate beneficial newborn organisms increases. As expected, population growth rates also increase with the benefit to cost ratio (see b–e).Population growth rates decrease with increasing osts see a,b. Incratio see b–e.m on the reproductive strategy for N = 8. We found that it is robust for the conclusion: the optimal reproductive strategy is binary splitting with maximum maturity size. At high m and high k, the asymmetric binary-splitting strategy 7 + 1 invades other asymmetric binary-splitting ones . Since 7 + 1 can keep the largest number of B cells in a newborn organism, this organism could gain growth advantages for high cell-type switching probability. A higher cell-type switching probability increases the heterogeneity of organisms, especially for large organisms. This leads to fewer B cells in an organism—thus, the population growth rate generally decreases with increasing m; see electronic supplementary material, figure S2b. For some reproductive strategies, a change in the cell-type switching probability leads to complex effects on population growth . This is the outcome of several factors: the cell-type switching probability m, the contribution threshold k and the structure of the population. We take the reproductive strategy 1 + 1 + 1 as an example to discuss the potential reason: when k = 2, the population has a lower growth rate when m = 0.5 because organisms cannot reach the threshold during their growth.Additionally, we investigated the effect of varying . 3.3ni + nj can be uniquely optimal, where ni and nj are positive integers, and ni + nj ≤ N . With the combined effects of size and threshold, we found new optimal binary-splitting reproductive strategies that are neither optimal in the effects of single perturbation only nor for thresholds only, including 2 + 2, 3 + 2, 4 + 2, 5 + 2, 3 + 3 and 4 + 3. Furthermore, under a beneficial size perturbation, we found n + 1  can be optimal both at small and large contribution threshold k . This is due to the fact that the threshold effects lead to a similar performance of reproductive strategies either at small k and at large k (b). Therefore, for combined size and threshold effects, the size effect has a larger impact on the performance of reproductive strategies . Consequently, the reproductive strategy n + 1 becomes optimal under an advantageous perturbation, where n = 1, …, 7. Newly emerged binary-splitting reproductive strategies have advantages for intermediate contribution thresholds k, suggesting that it is an outcome of the trade-off between the effect of size and threshold. For an adverse size perturbation, we found that the reproductive strategy n + 1 cannot be optimal (b), because the adverse size perturbation leads to poor performance of reproductive strategies that are influenced by the perturbation (b and 4d). The reproductive strategy 7 + 1 is an exception to this rule, as the threshold effect strongly influences it at k = 7. The optimal reproductive strategies observed are those that can obtain growth benefits from threshold effects and avoid the disadvantages from the adverse size effect. For example, 3 + 3 outcompetes 4 + 4 for k = 2 when a disadvantageous size perturbation occurs at n = 7. Both strategies can obtain growth advantages from threshold effects. However, adverse size perturbation decreases the population growth rate of 4 + 4, but it has no impact on 3 + 3. Thus the performance of reproductive strategies is the outcome of the trade-off between the effects of size and threshold. Our results suggest that all binary-splitting reproductive strategies can evolve under an appropriate choice of size effects (at a single size) and threshold effects.Finally, we investigate the optimal reproductive strategies under size and threshold effects combined. For simplicity, we only consider the size effects in the form of a single perturbation. All binary-splitting reproductive strategies + nj ≤ N a,b. Withld k see a,b. This large k b. Therefgies see a,c. Cons optimal b, becausurbation b and 4d.. 4Numerous reproductive strategies are conceivable for multicellular organisms, but only recently more attention has been paid to the evolution of such reproductive strategies ,20,41,42n + 1 resembles organisms passing through a unicellular phase, which is a striking feature for most multicellular organisms [n + 1 can evolve under size or threshold effects. This indicates an alternative way for evolving organisms with a single cell bottleneck. Our models also show the conditions where multiple reproductive strategies can evolve simultaneously. Each reproductive strategy can evolve when the size effects are the same for each cell size (χn are the same) or when the threshold is low . Many reproductive strategies have beneficial newborn organisms (nB ≥ k) when the threshold is low, leading to a large population growth rate. This result indicates that multiple reproductive strategies can coexist for an organism. Notably, the results resonate with the observation that one species can possess several reproductive strategies simultaneously in nature [The reproductive strategy rganisms . Previourganisms ,17. Inten nature ,15,45,46χn), we found the frequently observed optimal reproductive strategy 1 + 1. The finding suggests that 1 + 1 is the best reproductive strategy unless explicit advantages exist for a larger organism. The size effects considered in our model are functions of cell numbers rather than the task allocation of each cell. Previous work on the division of labour has instead focused on continuous functions [In our model, we can choose a flexible impact of size on organism growth. We investigated the size effects by first investigating the effects of single size perturbations. Although single size perturbations seem unrealistic for organism growth in nature, they provide a theoretically convenient approach towards understanding size effects. Size effects that are present by combing single size perturbations could have positive, negative or neutral effects on growth at each cell increment. The model assumption corresponds to studies concerning size effect on growth –34. The unctions ,48–50.We delineated the threshold effect of cellular interactions in a multiplayer volunteer game given the utility of game theory in depicting biological interactions ranging from social foraging to cancer development –58. We dm = 0.01. This focus assumed that cell-type switching is uncommon and mostly happens under environmental pressure in nature [nB ≥ k) ) newborn organisms, then homogeneous beneficial  newborn organisms dominate the population. Although heterogeneous beneficial newborn organisms grow fastest, they are not most abundant. First, heterogeneous beneficial newborn organisms do not produce themselves fast after reproduction. The reason is that A cells have higher payoffs; thus, they produce more A cells during the growth of heterogeneous beneficial newborn organisms. When the organisms reproduce, they are unlikely to produce more than one heterogeneous beneficial newborn organism. Second, although heterogeneous beneficial newborn organisms can be replenished from the reproduction of beneficial newborn organisms (via cell-type switching), the replenishment is slow as beneficial newborn organisms have lower growth advantages. Thus, it is also theoretically intriguing to investigate reproductive strategies under high cell-type switching probability. Such an investigation shows that the conclusion is robust across cell-type switching probability parameter space: the optimal reproductive strategy is the binary-splitting one with maximum maturity size . Although high cell-type switching probability mainly decreases the population growth rate, nevertheless it does not affect the relative performance of reproductive strategies. Within these binary-splitting strategies, the binary-splitting reproductive strategy producing the largest newborn invades others at high cell-type switching probability.We mainly investigated reproductive strategies with a small cell-type switching probability, n nature ,60. The Stanieria, Pleurocapsa and Dermocarpella [Our model investigated the competition of reproductive strategies via the growth rates of isolated populations. In many situations, this correlates with the ability to invade other populations—simply by outgrowing existing types. We can extend the model to a density-dependent population consisting of organisms with different reproductive strategies. Given the size and threshold effects, the invasive strategies would reflect the reproductive strategies observed in our model. Furthermore, our model considered the size effect via the number of cells in an organism, so the size function is discrete. In nature, some unicellular organisms grow by enlarging themselves rather than by cell divisions, such as carpella . Insteadructures ,62.