Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period.Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples ( Classical oncological therapies are often gonadotoxic and cause permanent infertility due to the depletion of the spermatogonial stem cell (SSC) pool, and damage of the testicular somatic environment ,2. More Transplantation of hematopoietic stem cells (HSCT) is a treatment option for a variety of inborn hematologic, immunologic, and metabolic diseases. While offering curative treatment for these diseases, HSCT usually requires myeloablative conditioning with high-dose chemotherapy, particularly by using alkylating agents. The risk of infertility later in life plays an important role in the decision of parents to perform HSCT on these children . Due to The SSCs, a subpopulation of the undifferentiated spermatogonia (SPG), are the earliest precursors of spermatids within the male adult. During testis development, SPG arise from the gonocytes by migration from the center to the periphery within the testis chords . The SPGFertility preservation refers to the freezing of germ cells or of gonadal tissue in the case of a given risk of infertility for the patient due to disease, medical treatment, age, or other influences . CryopreStarting from 2017, a few studies on human prepubertal testicular tissue using organ culture with the aim of propagating human SSCs and/or human in vitro-spermatogenesis have been published ,23,24,25This study intended to evaluate organ culture conditions initially tested on adult testicular tissue, with respect to their applicability to prepubertal testis tissue. For this purpose, tissues from three boys, aged 7, 9, and 12 years, collected prior to gonadotoxic therapy, which all exhibited serum testosterone below the detection level and SPG as the most advanced germ cells, were cultured using the gas-liquid interphase method in combination with StemFlex medium without or with additive components including colony stimulating factor 1 (CSF1), gonadotropins, activin A, and nodal. Tissue analysis, before, during, and after the culture, was performed on the protein, morphological, and ultrastructural level using immunofluorescence staining, semi-thin sections, and transmission electron microscopy (TEM). The used organ culture medium was analyzed in a close-meshed rhythm (every 2nd day) with regard to its hormone steroid profile by liquid chromatography-tandem mass spectrometry (LC-MS/MS), involving the determination of testosterone, progesterone, 17-hydroxyprogesterone (17-OHP), androstenedione, and estradiol. In addition to the organ culture approach, uncultured tissues from three additional boys of the same age groups , also admitted for fertility preservation, were examined for their number of DEAD-box helicase 4 (DDX4)-positive germ cells and piwi like RNA-mediated gene silencing 4 (PIWIL4)-positive SPG/SSCs, reflecting the heterogeneity of the tissues in the group of patients whose testis samples are frozen in the fertility preservation programs. To demonstrate the comparability of organ culture conditions between prepubertal tissues above a certain age of patients and adult testicular specimens at the ultrastructural level, mitochondrial sizes of undifferentiated SPG were determined in eight prepubertal and two adult testicular tissues using TEM micrographs.Here, we show that organ culture conditions developed on adult human testicular tissue are transferable to prepubertal patients 7 years and older, in regard to SPG maintenance and proliferation, which possibly can be explained by a comparability of the mitochondrial sizes in undifferentiated SPG and therefore a similar metabolism. Younger children, in contrast, displayed highly significant, larger mitochondria (MT), indicating a different mechanism of energy production. During the organ culture, Leydig cell (LC) functionality was evidenced by male steroid hormone production.For the organ culture experiments, prepubertal testicular tissue was acquired from three prepubertal patients of the University Hospital Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology, in the course of a biopsy for fertility preservation prior to a gonadotoxic therapy  ,30, wereAn informed and written consent for these studies, as well as an ethical committee approval  were obtained. All experiments were performed according to the Declaration of Helsinki’s ethical principles.For diagnostic purposes, one small testis fragment (~30 mg) from each patient was immediately transferred and fixed in 5.5% glutaraldehyde in phosphate buffer (pH 7.4) for a minimum of 3 h at 4 °C, and post-fixed for 2 h at room temperature in 1% osmium tetroxide in 0.1 M sucrose phosphate buffer . EmbeddiFor TEM, ultrathin sections (80 nm) were prepared and double-contrasted in 1% uranyl acetate for 30–60 min and Reynolds lead citrate for 3 min. Samples were photographed using a CM100 transmission electron microscope , equipped with a Quemesa Olympus TEM CCD camera .3 to 4 mm3, depending on the size of the biopsy. One small bit of each sample was directly fixed in modified Davidson fluid (mDF) for 1.5 h at 5 °C  and soaked overnight. After placing 1–2 testicular fragments on the gel stands using tweezers, complete media changes were performed on day 1. During the rest of the cultivation period, every second day 400 µL were removed and replaced with 500 µL fresh pre-incubated medium, keeping the agarose 4/5 submerged considering evaporation. The replaced media were frozen for later diagnostics.The other fragments were cultured in a modified testicular explant culture system established by Sato et al. (2011) at 35 °C and 5% CO2 ,28,32. FOn days 7, 14, and 21, one to two fragments per patient and condition were retrieved quickly, keeping the time outside the incubator as short as possible. Samples were fixed in mDF as earlier described and embedded in paraffin.Sections of 5 µm thickness were de-paraffinized in xylene and rehydrated through a descending alcohol series. For the assessment of the general histology of the cultured tissues, Mayer’s Hematoxylin was pipetted and removed after 5 min. Subsequently, the slides were submerged in tap water and blued for 3 min.For immunofluorescence staining, heat-induced antigen retrieval was performed on using 0.05 M boric acid at pH 8.5 in a microwave oven. After washing with TBS and permeabilizing with TBS + 0.1% Tween, sections were blocked for 1 h using TBS containing 0.1% Tween and 2% normal goat serum. Afterwards, primary antibodies  were appAs cell type-specific markers, DDX4 (germ cells), undifferentiated embryonic cell transcription factor 1 , PIWIL4 (SPG/SSCs), boule homolog, RNA binding protein ), nuclear SRY-box transcription factor 9 ), claudin 11  component), alpha-smooth muscle actin ), laminin alpha 1 , steroidogenic acute regulatory protein , cytochrome P450 family 17 subfamily A member 1 , and insulin like 3  were applied. For determination of germ cell proliferation, Ki-67 (MKI67) was employed in combination with the germ cell-specific markers.Between two and four tissue sections were examined manually per cell culture condition and week. Using the images taken on an Axio Vert.A1 microscope equipped with a Colibri 7 LED and an Axiocam 503 color camera, surface areas of the sections were measured with the Zeiss Zen 2.0 software . Cells were excluded if there was no DAPI counterstaining and their size and form did not fit the corresponding cell type, e.g., SPG, and they did not show the typical staining for the respective marker. For LAMA1 quantification, tubules were counted as LAMA1+, if at least 3/4 of the basement membrane showed the cell-specific marker expression.For the detection of dead cells/areas in the tissue, TUNEL staining was performed using the DeadEndTM Fluorometric TUNEL System . For the protocol, rehydrated sections as for immunofluorescence staining, were used. These were further processed according to the manufacturer’s protocol. Cells were counterstained with DAPI  and mounted with glass coverslips. Negative controls were performed without the rTdT enzyme; positive controls were treated with 1500 U/mL DNase1. Imaging was done with an Axio Vert.A1 equipped with a Colibri 7 LED and an Axiocam 503 color camera. For quantification, the channel that represented TUNEL was separated, and the area was estimated using an intensity threshold in ImageJ . Subsequently, the area of the whole section was measured, and the percentage of the area containing cells with fragmented DNA was calculated. Initially, ELISA assays were performed to determine testosterone levels in organ culture supernatants using the Testosterone Human ELISA Kit ab174569 . Assays were carried out according to the manufacturer’s protocol. Briefly, a calibrator, samples, and positive controls were dispensed into the wells. HRP enzyme conjugate was added, mixed thoroughly, and incubated for 60 min at room temperature on a plate shaker. After striking out the contents of the wells and washing, TMB substrate solution was added, and the plate was incubated for 15 min. The reaction was stopped with a stop solution, and absorbance of each well was determined at 450 ± 10 nm using an Anthos HT3 microplate reader . The results have been calculated automatically via a 4 PL (4 Parameter Logistics) curve fit. Samples with a concentration higher than the range of the calibrator have been diluted. Serum from a 25-year-old healthy male was used as a control.Due to an unsuitable standard concentration series for the level of testosterone concentration to be determined in the organ culture supernatant of the prepubertal samples, and thus a pre-programmed measurement inaccuracy using the ELISA assay, samples from the patients with measured absorbance values were referred to liquid chromatography-tandem mass spectrometry (LC-MS/MS) (see next section).Steroid hormones in organ culture supernatants were determined at the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig . A 100 µL of supernatants were used for the simultaneous quantification of 17-OHP, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC–MS/MS as previously described . Lower lt-test with Bonferroni multiple-comparison correction.For these investigations, TEM micrographs were used. Photos were imported into the Zeiss Axiovision software . For correct scaling, the length of the scale bar in pixels was manually measured. Subsequently, the surface areas of the MT were manually determined using the spline function. To exclude a bias due to the differential weighting of cells with many MT and cells with only a few MT, the mitochondrial sizes of the undifferentiated SPG were surveyed in a cell-independent manner. In total, 1023 MT of prepubertal SPG and 428 MT of adult SPG were determined. Statistics were calculated using a pairwise t-test with Bonferroni multiple-comparison correction  )  starting from the native tissue up to cultivation week 3 with partly significant and partly non-significant statistical values a,b. In tnditions a. Roughlons (2]) a, the DDons (2]) b. It is ons (2]) a,b, cond2 , the 9-year-old patient with SCD displayed the lowest number with 71 cells/mm2 g, the 7-2  was displayed. These findings are discussed in At first glance and with respect to the ranking, there seems to be no direct dependence between the number of DDX4-positive and PIWIL4-positive cells in the native tissues of the patients studied here. Nevertheless, upon closer inspection, an interesting pattern appears: Firstly, in the case of the two 7-year-old patients with ß-thalassemia major, the proportion of the total number of DDX4-positive cells was approximately 100 cells/mmIn both experiments, few positive BOULE signals were detectable after the first week and even in one sample after day 14 . InteresIn the native tissues of both patients, nuclear SOX9 expression as well as cytoplasmic expression of the BTB component CLDN11 was present, signaling a certain and approximately concordant maturation status of the SCs in the two tissues cultured over three weeks . During To further elucidate the maturation status of the prepubertal SCs at the ultrastructural level, we investigated the native tissues of the 7- and 12-year-old boys by TEM. Indeed, it could be seen that the SC nuclei often appeared in a more immature form, namely, more rounded and less lobulated compared to adult SCs. They also presented a less prominent nucleolus a,b. In cTo study cellular functionality and secretory competence of the LCs in the used organ culture medium, testosterone was initially analyzed for the 7-year-old boy and the 9-year-old SCD patient, applying an ELISA assay. Here, measurable values were only obtained for the 7-year-old patient, whereas absorbance remained at zero when testosterone in the culture medium of the 9-year-old SCD patient was investigated. Due to a difficulty with the ELISA method, it turned out that the standards included in the assay did not produce a calibration curve suitable for the value range of the prepubertal samples, so accurate calculation could not be reached utilizing this approach. As an alternative strategy, LC-MS/MS was applied with the additional advantage of simultaneous determination of testosterone and a variety of other steroid hormones. In In the adult testis, testosterone and precursor hormones are synthesized by the LCs. In this process, progesterone, which itself constitutes the precursor for 17-OHP, is produced from pregnenolone. In turn, the 17-OHP represents an intermediate in the synthesis of several hormones such as cortisol, estrogen, and testosterone. Within the testosterone production pathway, it is first converted into androstenedione, which is then either transformed into testosterone or metabolized by the CYP19A1 aromatase into estrone, the precursor for estradiol. Testosterone may also be converted to estradiol by CYP19A1 aromatase ,52.Organ culture experiments on the testis tissue of the 7-year-old patient and the 12-year-old XIAP deficiency patient revealed that under the selected culture conditions, LCs are capable of producing testosterone and its precursor hormones over the entire cultivation period , with thWhen comparing the three different culture conditions, patient-specific differences come to light. In the case of the 7-year-old patient, highest hormone values were detected in condition C, while the lowest were detected in condition A  conditions and time points. This might explain the high progesterone peak under condition A at day 15 d and theFor further identification of the LCs in the cultured tissues, two tissue sections from the 12-year-old patient, cultured for 3 weeks (conditions B and C), were examined using semithin sections. As a result, LCs could be detected in both pieces of the tissue. Based on the transferability of SPG- and organ-culture-conditions from adult to prepubertal tissue, regarding patients of a certain age group demonstrated by our experiments, and therefore to compare key morphological characteristics important for the metabolism of SPG on the ultrastructural level, we used TEM to examine the mitochondrial size of prepubertal SPG compared to adult A-SPG. For this purpose, eight prepubertal testicular tissues from boys of different ages (between 1.5 and 13 years) and two tissues from adult males, showing complete spermatogenesis see , were set-test plus Bonferroni correction revealed that on average, the MT in the SPG of the two younger patients (1.5 and 5 years old) were of significantly larger size  and StemPro®-34 SFM   as well as different diseases were cultured under three different conditions based on the non-xeno-free StemFlex culture medium, which we had found to be the most favorable culture medium for adult human organ culture in comparison to NutristemNY, USA) . StemFleActivin A and nodal represent pleiotropic growth factors of the TGFß superfamily. By binding to the same receptors, their ligands signal via the same pathway, resulting in the activation of the SMAD transcription factor family, which in turn is implicated in downstream actions such as cellular stemness, proliferation, and cell cycle regulation ,62,63. IThe effects of the organ culture conditions on the individual cell populations over time are summarized in 2). In our case, we quantified the SPG in the tubular areas of the tissue pieces and measured in “cell count/mm2”. Therewith, the cell number is related to the whole tissue piece and only fibrous areas are excluded. In this way, the quality of the entire cultured sample is considered, which is not taken into account when, e.g., only the “well-preserved tubules” are determined [Within our organ culture approach, DDX4-positive germ cells and PIWIL4-positive SPG could be maintained throughout the entire cultivation period in the tissue of the 7-year-old and the 12-year-old patient . Neverthtermined ,21. As atermined ,80,81. Ctermined . Interestermined   . MedranoComparing our three organ culture conditions in terms of number of the germ cells and SPG maintained over the cultivation period, condition C performed best in 9 out of 12 cases. However, statistical significance could not be demonstrated, which most likely results from the low number of samples used in this study. Due to the small sample size, these results should be regarded with caution.Testicular somatic cells include SCs, LCs, and PTCs. While the SCs constitute a fundamental component of the SSC niche, the interstitial LCs are responsible for male steroid hormone secretion. The PTCs, on the other hand, enclose the outside of the seminiferous tubule basement membrane and represent the production site for the extracellular matrix proteins . During 2; For the PTCs, the markers LAMA1 and alpha-SMA were employed. According to the study by Kurek et al. , the preFor the assessment of the maturation status of the prepubertal tissue and as a standard marker for the testicular PTCs, alpha-SMA was utilized. Alpha-SMA represents an excellent marker for assessing the prepubertal status of testicular tissues in the non-human primate species marmoset, and partial expression of this protein marks an ongoing process of PTC differentiation, which is still incomplete ,39. BefoThe markers SOX9 and CLDN11 were applied to assess the maturation status of the SCs. While SOX9 is expressed in mature SCs and also in the adult human testis, CLDN11 represents a component of the BTB, which is necessary for SC tight junction formation . CLDN11,LC maturation over the culture period was determined using various cell type-specific markers. STAR could be detected in the native tissues of both patients, whereas CYP17A1 was also present in the tissues of both patients, but to a much weaker extent in the case of the 7-year-old patient. INSL3 was only seen in the tissue of the 12-year-old patient, and here there is great heterogeneity in terms of the number of the INSL3 signals. During the organ culture, tissues from both patients showed an increase in CYP17A1 protein expression, although this was much more pronounced in the case of the 12-year-old patient. In the tissue of the 7-year-old patient, the strongest CYP17A1 signals could be detected in condition C. The situation was similar with regard to the marker INSL3: The cultured tissue from the 12-year-old boy displayed INSL3 signals in all areas, whereas in the tissue of the 7-year-old boy, INSL3 protein expression was seen only at a few sites. The presence of the LCs in the tissue cultured for three weeks was simultaneously demonstrated by means of semithin sections and at the ultrastructural level . HoweverIn addition to the examination of the LCs at the morphological and protein expression level, steroid hormone production was quantified in the organ culture supernatant by LC-MS/MS as a functional proof. Here, the concentration of testosterone and its precursor hormones, progesterone, 17-OHP, and androstenedione, as well as estradiol, was measured. Overall, the steroid synthesis and conversion validated the endocrine function of the tissues in our organ culture approach. In both of the patient samples that were used for the organ culture experiments, a dynamic in hormonal secretion is clearly visible. The detailed breakdown of steroids enables us to retrace steroid enzyme function in the testis. In Estradiol, which is also included in our mass spectrometry, is synthesized by converting testosterone via aromatase. In almost all cases, high estradiol levels were observed with high testosterone. Sharma et al. (2022) reported similar results comparing high-dose and low-dose exposition to human chorionic gonadotropin (hCG) and FSH in a marmoset microfluidic setup . In our The higher testosterone secretion in condition C compared to condition B, even though only tissue from a few patients could be examined, fits with the data of the Pollard study which showed that mice testicular macrophage depletion, and thus a CSF1 absence, leads to a reduction in the testosterone levels .In our study, the stimulability and thus functionality of the LCs was demonstrated by the determination of the steroid hormone profile by LC-MS/MS. This is the first study that beside testosterone secretion, also considers the production of the testosterone precursor hormones in human prepubertal testicular organ culture.2  and those which were further examined (2nd part of the study), were found to be highly heterogeneous with respect to the germ cell (DDX4+) and spermatogonial cell (PIWIL4+) quantities: The total DDX4+ cell counts varied between approximately 70 and 1050 cells/mm2 g, whereb2, see the penultimate pink bar in Calculating the differences between the amounts of DDX4+ cells g and PIWSince the 1st part of our study showed the feasibility of organ culture conditions developed on human adult testicular tissue, for prepubertal testis tissue from patients 7 years of age and older, with respect to SPG/SSCs preservation, we aimed on the identification of similarities in the ultrastructure of the MT from adult and prepubertal undifferentiated SPG. MT represent the key components in the metabolism of a cell, and a recent study  demonstrThese findings indicate that different culture conditions regarding oxygen concentrations might be useful for the in vitro-cultivation of prepubertal human SSCs from patients of different ages. Further research on this topic, including large-scale studies closely covering the entire male prepubertal age range and single cell analyses, could be helpful to complement the data presented here.The culture conditions used in this study promote SPG maintenance and proliferation as well as the maturation of the prepubertal testicular somatic cells. The LCs, furthermore, could be stimulated for testosterone and precursor hormone production. FSH, LH, and growth factors, especially CSF1, exerted positive effects on the cultured cells, although due to the small number of samples applied, no significant effect could be reported. Due to the small sample size, the results from the cultivation experiments should be viewed with caution. Further optimization in regard to the cultivation of SPG and SSCs in boys aged seven years and older might be achieved by application of the culture conditions under hypoxia, as indicated by the ultrastructure of MT in the undifferentiated SPG of patients from this age group.


This historical review aimed to summarize the main changes that colorectal carcinoma (CRC) staging systems suffered over time, starting from the creation of the classical Duke’s classification, modified Astler–Coller staging, internationally used TNM  staging system, and ending with molecular classifications and epithelial–mesenchymal transition (EMT) concept. Besides currently used staging parameters, this paper briefly presents the author’s contribution in creating an immunohistochemical (IHC)-based molecular classification of CRC. It refers to the identification of three molecular groups of CRCs  based on the IHC markers E-cadherin, β-catenin, maspin, and vimentin. Maspin is a novel IHC antibody helpful for tumor budding assessment, which role depends on its subcellular localization (cytoplasm vs. nuclei). The long road of updating the staging criteria for CRC has not come to an end. The newest prognostic biomarkers, aimed to be included in the molecular classifications, exert predictive roles, and become more and more important for targeted therapy decisions. Colorectal cancer, being mostly colorectal carcinomas (CRC), represents the third most diagnosed cancer and the second cause of cancer-related death . Over thThe current review is focused on the CRC staging systems, from the classical Dukes’ classification to the TNM  stages included in the latest published edition of the American Joint Committee of Cancer (AJCC), with continuous updates over the past decades, together with relevant prognostic markers used by recent studies in the search for molecular classification of CRC, including our team’s contribution to the field.For this review of internationally used staging systems, peer-reviewed publications identified on PubMed, Scopus and web of science databases using as keywords colorectal carcinoma AND “staging”, “staging system Dukes”, “staging Astler Coller”, “staging system AJCC”, and “TNM” or “molecular classification” were included. The databases were searched from inception to 4 July 2022.https://cancerstaging.org/references-tools/deskreferences/pages/default.aspx (accessed on 3 July 2022). After the title’s evaluation, the selection required checking the abstracts and, in the end, the full-text variant of the articles was read. Only articles written in the English language, based on human tissue studies, with available abstract and full text, were taken into consideration. Staging-related data were also extracted from older versions of the AJCC manuals available online at After deduplication, the initial search resulted in 659 papers. After removing letters to the editor, and articles with unavailable full text in English, a number of 286 papers was selected for full-text screening and 66 of them were included in the present review.Staging systems are used to enable the prediction of survival, an internationally appropriate and uniform case evaluation, and treatment decision. Dukes proposed in 1932 the first staging system of CRC, starting with the rectum and then for colorectal segments ,6. The fFurther subclassification and modification of these stages were conducted by Astler and Coller (MAC) in 1954. They proposed splitting former stage A into A and B1. Only superficial tumors limited to the mucosa were included in stage A and those infiltrating the submucosa, but not crossing muscularis propria in stage B1. Ex-stage B becomes B2. No metastases were included in stages A, B1 and B2. Cases with positive LNs were included in C stages, respectively, C1 and C2, corresponding to B1 and B2 with associated LN metastases  7,8,9].,8,9.7,8,Turnbull proposed in 1967 a new stage—stage D—for tumors with distant systemic spread or direct invasion of the peritoneum—which is partially equivalent to the TNM stage IV introduced in 1977  and keptAlthough new parameters and sub-divisions were further included in the TNM staging system, the Dukes paradigm of considering lymph nodes as one of the strongest prognostic parameter is kept even in the 8th edition of the AJCC Manual .From 1977 until nowadays, the well-known and internationally utilized pathological TNM (pTNM) staging suffered periodical changes which are included in well-known AJCC manuals . ContinuThe most significant changes were added starting with the 6th edition of AJCC in 2002. Although the T, N, and M parameters were not modified, following the Dukes paradigm, cases were sub-classified based on the number of metastatic nodes and the clinical stages I, II and III was suggested to be included in the histopathological reports . For theProblems regarding the influence of pre-operative therapy were also firstly addressed in the 6th edition of AJCC with the addition of the letter “y” in front of the pTNM stage, with no other evaluation differences compared to untreated tumors . PreciseAnother useful aspect refers to the presence of multiple synchronous colorectal tumors. It is represented by the symbol “(m)” inserted at the end of the pTNM stage .In the last two editions of the AJCC manuals, it was included in the stage N1c for cases with the presence of tumor deposits in the absence of LN metastases (7th edition), based on a more unfavorable prognosis compared with N0 staged cases. A distinct M1c stage was proposed for the presence of peritoneal carcinomatosis (8th edition). It was based on the worse outcome of these cases compared with those spreading in other organs. The T4 stage was also sub-divided into T4a and T4b  11,16,2,21,22.Currently, it is indicated to include in the histopathological reports, along with the TNM stage criteria , those pAs the systemic inflammatory response (SIR) plays role in carcinogenesis, more and more studies are focused on the prognostic role of the SIR-related parameters such as neutrophil-to-lymphocytes ratio (NLR) or lymphocytes-to-monocytes ratio (LMR). LMR represents the ratio between preoperative lymphocyte and monocyte counts assessed at baseline. LMR and NLR values are correlated with the TNM stage. High preo-operative NLR (over 3.11) and low LMR are indicators of poorer overall survival rates ,25.Rectal cancer accounts for one-third of all CRCs. There are some aspects that regard this segment only. Starting with the last (8th) edition of AJCC, it is recommended, for rectal carcinomas, to evaluate the quality of mesorectal fascia ,11,14. TPreoperative imagistic evaluation of CRC can be performed with computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) combined with CT (PET/CT). They have advantages and limitations. CT is useful for lymph node assessment. MRI is mainly used for checking a suspicion of relapses, especially for rectal tumors and suspected hepatic metastases. PET/CT is indicated to evaluate the whole body and check distant metastases .Proper staging of CRC requires a precise evaluation of LN status. It should be performed by a transdisciplinary team and start before surgery with CT or MRI scans. The suspect LNs are evaluated based on imagistic criteria such as size , shape, contour, and heterogeneity . AfterwaFor rectal cancers, pelvic MRI is the gold standard imaging way for evaluation of primary tumors and local recurrences, even for cases with a large post-radiotherapy fibrotic scar. The risk of local relapse can be predicted based on the distance between the tumor and circumferential resection margins, combined with the presence or absence of extramural invasion .Further changes and ways of perfecting the evaluation methods as a response to current challenges in the diagnostic and therapeutical case management are continuously being studied and new valid evidence-based results will help future staging updates ,33,34.For a proper MRI evaluation of the TME, the newest classification beyond TME (BTME) was recently proposed. After pelvic MRI, cases can be grouped based on their localization in the eight compartments: 1. Anterior above peritoneal reflection ; 2. Anterior below Peritoneal Reflection (Genitourinary organs and pubic symphysis); 3. Central ; 4. Posterior ; 5. Lateral ; 6. Infralevator ; 7. Anterior urogenital . The worse survival was reported for patients with tumors located in the first compartment  same as for those with tumors involving multiple compartments .[18F]-FDG PET combined with MR ([18F]-FDG PET/MR) was recently proved to have high specificity and sensitivity for the diagnosis of CRC, evaluation of the free margins (distance from tumors) and identification of distant metastases. Due to limited spatial resolution, the preoperative T stage cannot be properly performed with PET/CT. High specificity, but low sensitivity was also proved for N staging .Differences in tumor behavior and response to therapy in same-stage CRC cases have increased the need for gene-expression studies and the creation of a molecular classification that would facilitate targeted therapy ,37,38,39Tumors belonging to the CMS1 subtype are hypermutated, with BRAF mutant status, microsatellite instability (MSI-H), and an important immune reaction. CMS1 group is also known as MSI immune. Carcinogenesis seems to be driven via JAK-STAT and PD-1 signaling pathways ,40. AlthCMS2  and CMS3 (metabolic) represent epithelial subtypes. CMS2 is chromosomally unstable, with activation of WNT and MYC signaling pathways. CMS3 shows metabolic deregulations and KRAS mutations and comprises MSI-H and one-third of cases that are microsatellite stable (MSS). The CMS3 MSS-carcinomas are architecturally such as MSI tumors ,39,40,41CRCs with stromal invasion, angiogenesis, and transforming growth factor β (TGF-ß) activation are included in the CMS4 subtype, which is also known as the mesenchymal subtype ,39,40,41In one of the recent studies, a refined classification of the CMS2 and CMS3  was proposed based on intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F). It was called “iCMS” or “IMF” classification but implementation in daily practice is not easy to be performed .Studies confirm response and outcome differences between tumors included in the four CMSs, larger cohorts being required for any valid official changes ,45,46,47Heterogenous research results indicate better overall survival (OS) for CRCs CMS2 and CMS3 when bevacizumab or cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is associated with classical therapy ,44,45. TBesides aiding the molecular classification process, microsatellite status by itself shows important diagnostic and therapeutic implications. It represents the presence of repeated sequences encompassing 1-6 nucleotides, causing mutations of the DNA mismatch repair (MMR) genes , mutations that can be inherited (Lynch syndrome) or developed sporadically ,37. ScreMultiple studies attempted to molecularly classify CRC using the expression of IHC antibodies for legit reasons such as cost-efficiency and availability in most pathology departments ,50,51,52These stains were not enough for the distinction between CMS2 and CMS3, which are mainly driven via the Wnt pathway . Li X. eOur team also focused on classifying CRC based on IHC reactions and used markers of EMT such as E-cadherin, β- catenin, vimentin, and maspin, evaluated in both tumor center and invasion front/tumor buds  51,54,5,554,55. Tumor budding, defined as the single tumor cells or groups of no more than four tumor cells identified in the invasion front, represents an extensively studied parameter with a Hematoxylin-Eosin +/− cytokeratin slide-based evaluation protocol published in 2017, represents an independent prognostic marker not yet included in AJCC staging manual, but its importance and suggestion for addition in the pathological report are mentioned in oncological practice guidelines for both colon and rectal carcinomas ,58,59,60Evaluation of subcellular maspin’s expression, combined with microsatellite status, could also be of therapeutic relevance ,64. CytoProved to be related to EMT and tumor-associated angiogenesis, maspin is opening a window for potential targeted therapy ,65,66,67Like other tumors, it is thought that, in the near future, the therapy of CRC will be completely based on molecular diagnostic tests. Deep learning machines can already be used for the evaluation of whole slide images and establishing histological grade, budding degree or other prognostic parameters .The role of pathologist needs to be revisited and next-generation sequencing platforms will replace large parts of ancillary tests. However, as most of the molecular tests are performed from paraffin-embedded tissues, the tissue quality still depends on the pre-analytical processing. Identification of the tumor-rich areas also depends on the pathologist and its role remains crucial for proper staging and lymph node harvesting .This review shorty presented the historical evolution of CRC staging systems, using detailed tables to highlight the main modifications and the current interest in molecular classification. It aims for better stratification of cases, above classical staging limitations, in constant search for prognostic biomarkers with beneficial therapeutic impact. Promising discoveries have been made, but further studies are necessary to validate these achievements and include them in future staging manuals.


The studies also revealed increased levels of renal blood flow (RBF) in stenotic kidney (STK)  and the glomerular filtration rate (GFR) of STK . In clinical studies, the cortical perfusion and fractional hypoxia of the contralateral kidney (CLK) were alleviated by MSC therapy. Taken together, this meta-analysis revealed that MSCs therapy might be a promising treatment for RAS. However, due to the discrepancy between preclinical studies and early clinical trials outcomes, MSC therapy couldn’t be recommended in clinical care for the moment, more high-quality randomized controlled clinical trials are needed to validate our conclusions and standardize MSCs protocols.Renal artery stenosis (RAS) causes severe renovascular hypertension, worsening kidney function, and increased cardiovascular morbidity. According to recent studies, mesenchymal stem cells (MSCs) administration is a promising therapy for the improvement of RAS outcomes. The meta-analysis aims to evaluate the therapeutic effects of MSC therapy on RAS. We performed a search in MEDLINE, Web of Science, Embase, and Cochrane Library from inception to 5, October 2022. We included 16 preclinical and 3 clinical studies in this meta-analysis. In preclinical studies, the pooled results indicated that animals treated with MSCs had lower levels of systolic blood pressure (SBP) (SMD = − 1.019, 95% CI − 1.434 to − 0.604, I RAS prevalence is approximately 1–3% of the population with hypertension and up to 20% of the population with secondary hypertension4. Kwon et al. showed that RAS occurred more frequently in hypertensive patients aged 65 years or older5. RAS increases hypertension and post-stenotic kidney damage6, leading to end-stage renal disease, more specifically in elderly patients. What’s more, patients with RAS have an increased risk for developing cardiovascular complications, increasing morbidity and mortality7. Conlon et al. reported that the presence of RAS in patients with coronary disease independently doubled the risk of mortality even when coronary revascularisation was performed8. In past decades, lipid lowering-drugs, antihypertensive drugs, and renal revascularization were used to treat RAS. However, two large clinical trials, ASTRAL and CORAL, demonstrated that renal revascularization had no advantage when compared to the medical treatment11. This might because not merely chronic hypoxia and reduced blood flow, but activation of the renin–angiotensin–aldosterone system, increased oxidative stress and cytokine release, microvascular dysfunction and rarefaction, and kidney fibrosis are involved in the underlying mechanisms. Conversely, recommendations of therapy were discordant in various guidelines. RAS patients suffering irreversible loss of kidney function and persistent hypertension usually develop into end-stage kidney disease (ESRD) and cardiovascular events, causing heavy economic burden for families and society, especially in developing countries. Therefore, it is necessary to develop a more effective alternative or adjunctive therapy for RAS treatment.Renal artery stenosis (RAS) is the primary cause of renovascular hypertension (RVH)12. Due to the capacity of their differentiation potential, self-renewal and immunomodulatory cytokine secretion, MSCs was widely studied in many diseases, including autoimmune disorders13, acute kidney injury14, chronic renal failure15, diabetic kidney disease16, and stroke17.Cell-based therapies attracted attention in numerous fields of medical research in recent years. Mesenchymal stromal cells (MSCs) are isolated from various adult tissues, such as bone marrow, adipose tissue, umbilical cord blood, and deciduous teethTo date, some studies regarding RAS indicate that the administration of MSCs can ameliorate the loss of renal blood flow, kidney function, as well as elevate the release of inflammatory factors. However, the efficacy of MSCs administration on RAS remains unclear. There is a reinforced need to conduct an overview that patients and clinicians can utilize. Thus, this systematic review and meta-analysis of preclinical and clinical studies aims to evaluate the efficacy of the MSCs treatment in RAS.We searched four online databases: MEDLINE, Embase, Cochrane Library, and Web of Science from up to October 5, 2022. Searched terms used in this article were listed as follows:  AND . Furthermore, reference lists were also reviewed for the possibility of additional literature. Two researchers independently screened the collected articles.Inclusion criteria of the eligible literature are as follows: (1) randomized controlled trials, comparative studies, or controlled trials involving animal models of RAS or patients with RAS, (2) animals or patients in the studies who received MSCs treatment, (3) studies that have a corresponding comparison group, (4) Data regarding MSC treatment or control groups should be provided in detail, (5) studies that provided efficacy outcomes .Exclusion criteria are as follows: (1) case reports, meeting abstracts, repeat studies, letters, reviews, or meta-analysis and studies where the full text was unavailable, (2) studies with insufficient data, (3) studies that focused on the treatment using other stem cells or agents.Two investigators independently reviewed the titles and abstracts according to inclusion and exclusion criteria. Once disagreements arose, a third investigator reviewed the articles and made the decision. Relevant data were recorded in a standardized form in Microsoft Excel. The preclinical form included the first author, year, location, species, the number of groups, type of MSC, modeling methods, the dosage of MSC, delivery, follow-up duration, and efficacy indicators , renal blood flow, serum creatinine (Scr), urine protein, body weight, and inflammatory markers). The clinical form included the first author, year, location, number of groups, type of MSC, modeling methods, the dosage of MSC, route of MSC delivery, endpoints, and follow-up duration. The data were extracted from graphics using Get Data Graph Digitizer 2.25 software for studies that did not supply direct results.18. For the clinical studies, we used the Methodological Index For Non-randomized Studies (MINORS) tool for the non-randomized controlled studies19. The GRADE assessment was also performed to assess the quality of the evidence. The quality assessment was carried out by two investigators independently, and a third investigator resolved any disagreements.For preclinical studies, the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool was employed for quality assessment20. The weighted mean difference (WMD) and standard mean difference (SMD) with 95% confidence intervals (CIs) were used for appropriate continuous variables. According to previous studies, we used the method below to choose the effect model. The effects of the outcomes were pooled using a fixed-effect model, while a random model was employed when significant heterogeneity was detected. Heterogeneity was assessed by I2 and considered significant when I2 > 50%23. Potential publication bias was assessed via Funnel plots, as well as the Bagger’s and Egger’s tests. P < 0.05 (two-sided) was considered statistically significant in our meta-analysis.This study followed the recommended PRISMA statement. STATA 12.0 statistical software package  was used for statistical analysis. All median with range or interquartile range were converted to the form mean with standard deviation39 and 3 clinical studies41 were included in our meta-analysis, in which one study performed both animals and human trials. If more than one experiment was carried out in a single study, we regarded each experiment as independent.Our study identified 2844 relevant studies initially, including clinical and preclinical studies. After removing the duplicates and screening the titles and abstracts, 65 studies were left. The remaining 65 studies were carefully reviewed and 47 were further excluded due to lack of data, being off-topic, and unavailability of full texts. The flowchart for the screening process of the eligible trials is shown in Fig. In 16 preclinical studies, 8 were conducted in pig models, 6 were conducted in rat models, and 2 were conducted in mouse models. The RAS or RVH model was induced using an irritant coil placed in one main renal artery. Additionally, a high-cholesterol diet was used in the atherosclerotic renal artery stenosis (ARAS) models. Different types of MSCs were used within the studies, adipose-derived stem cells (ADSCs) (n = 11) and bone marrow mesenchymal stem cells (BM-MSCs) (n = 5) were included. The characteristics of the included animal studies are listed in Table Three clinical studies, all are non-randomized controlled trials. A total of 84 patients were involved. The duration of follow-up was 3 months. A summary of the clinical studies characteristics is provided in Table The detailed information of the quality assessment within the preclinical studies is shown in Table 37 reported systolic blood pressure (SBP) levels. A fixed-effect model using Cohen’s analysis was employed to compare the levels between the MSC treatment and control groups. Compared to the control group, the SBP decreased significantly in the MSC treatment group    level was found in 9 studies39 evaluated the degree of RAS and the outcomes demonstrated that the MSC treatment group had an equal degree of RAS compared to the control group   ; no significant increase in renal blood flow was observed in the MSC treatment group . In addition, significantly increased cortical perfusion was measured in 2 studies36; however, increased cortical perfusion was not observed in the MSC treatment group .The level of renal blood flow (RBF) in stenotic kidney (STK) was assessed in eight preclinical studies37 measuring serum creatinine suggested that animals in the MSC group had a lower serum creatinine level  36.The pooled results of nine studies39. The blood samples were collected from the inferior vena cava. Compared to the control groups, the MSC administration groups had a lower level of PRA  .The plasma renin activity was measured in seven studies39. Compared to the control group, MSC administration preserved GFR of STK  33. In addition, creatinine clearance was assessed in two studies35; however, it was not significantly different between the MSC and control groups .Glomerular filtration rate (GFR) in STK was measured in ten animal studies37 included the assessment of urinary volume. There is no significant difference between the MSC and the control groups . Similarly, the urinary flow in STK (2 studies were included)33 had no significant response to MSC intervention . Nevertheless, a higher level of urinary sodium excretion (3 studies included)37 and a lower level of urinary potassium excretion (2 studies included)37 were observed in the MSC group .Two studies37. No significant difference between the MSC treatment and the control groups was observed  .Furthermore, the protein levels of renin, angiotensin-converting enzyme (ACE), and Ang II receptors types1 (AT 1) and 2 (AT 2) in STK were detected using western bolt in two studies39 assessed the level of body weight. The pooled outcomes were analyzed using a fixed-effect model. However, no apparent differences were found between the MSC and the control groups  . The pooled effects of MSC intervention on kidney weight of CLK were also detected in three studies. Similarly, the MSC group had an equal kidney weight of CLK compared with the control group . Besides, the volume of the cortex in STK was also measured in two studies29. The results indicated that the MSC group obtained a greater cortex volume in STK . However, one study showed that the volume of all STK was unaffected by MSC treatment (P > 0.05).The kidney weight of STK was evaluated in three preclinical studies39 evaluated the level of cortex microvascular density in STK using micro-CT. The pooled outcomes demonstrated that MSCs could improve cortical microvascular density in STK  . The number of capillaries per tubule in the cortex and medulla was measured in 3 studies39 showing that MSCs improve capillary density in the cortex and the medulla .Four studies30. Compared to the control group, total cholesterol in MSC group has no remarkable difference. . The levels of triglycerides, HDL (high density lipoprotein) and LDL (low density lipoprotein) were detected in two studies30. There was no significant difference in triglycerides and HDL between MSC group and control group, while MSC therapy can significantly decrease the level of LDL .Total cholesterol was detected in 3 studies39 and 339 studies, respectively. The results indicated that STK cortical and medullary hypoxia was not significantly alleviated by MSC intervention . Similarly, the MSC therapy do not improve tubular function in STK 29.Renal oxygenation and tubular function were evaluated using blood-oxygen-level-dependent magnetic resonance imaging (BOLD MRI). The cortical and medullary oxygenation was assessed in 226 for IFN-γ, two studies26 for TNF-α, three studies30 for IL-10, and two studies26 for MCP-1 were utilized in order to evaluate the efficacy of MSCs. The pooled outcomes showed that MSCs were effective for the net renal release of IFN-γ, TNF-α, IL-10, and MCP-1  , tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and monocyte chemoattractant protein-1 (MCP-1) were detected. Two studies28, TNF-α (7 studies included)38, IL-10 (4 studies included)29, MCP-1 (3 studies included)39, vascular endothelial growth factor (VEGF) (3 studies included)29, transforming growth factor-β (TGF-β) (2 studies included)36, tissue inhibitor of metalloproteinase-1 (TIMP-1) (2 studies included)38, and matrix metalloproteinase-2 (MMP-2) (2 studies included)38. In summary, the MSC group showed statistically significant decreases in the levels of IFN-γ, TNF-α, and MMP-2 as well as significant increases in the levels of VEGF and IL-10 . However, there was no significant difference between the MSC and the control groups regarding the levels of MCP-1, TGF-β, and TIMP-1 .The western bolt was used to measure the levels of IFN-γ (2 studies included)30 were included for the measurement of circulating levels of isoprostane. The pooled outcomes suggested that MSC intervention can reduce the circulating levels of isoprostane .Two studies39 and a picrosirius red staining method (3 studies included)37. Compared with the control group, the degree of renal fibrosis was alleviated by MSC treatment .In addition, two studies29 were included to assess the tubular injury score. The MSC group had a lower score than the control group . The percentage of glomerulosclerosis was analyzed in two studies28, and the results showed that MSC could improve glomerulosclerosis . The apoptosis and oxidative stress levels were assessed via TUNEL36 and DHE39, respectively .Renal fibrosis was evaluated using a trichrome staining method (6 studies included)40 were included for the assessment of SBP and DBP. No benefit of MSC intervention was observed in the MSC groups    Fig. E. Howeve41. We compared the changes from baseline to 3 months between the MSC and the medically treated groups. However, compared to the medical only treated groups, MSCs had no remarkable outcomes excepted within the cortical perfusion and fractional hypoxia of CLK   Figs. , S5, S6.The funnel plots and Egger’s tests showed significant publication bias in GFR of STK (P = 0.006) (Additional file In our meta-analysis, 16 preclinical and 3 clinical studies in 18 publications were included to evaluate the efficacy of MSC therapy for RAS. No adverse events were reported in the animal and the clinical researches. In the preclinical studies, significant differences were observed in the levels of SBP and RBF in STK, Scr, RRA, GFR, as well as in the microvascular density of the cortex. Besides, the levels of cytokines, including the net renal release of IFN-γ, TNF-α, IL-10, and MCP-1, as well as renal fibrosis, also responded to MSC therapy. The results suggest that MSC might be a potential therapeutic agent for RAS. However, similar efficacy of MSCs administration did not appear in clinical trials. Furthermore, only the cortical perfusion and fractional hypoxia of CLK were improved by MSC therapy, indicating an urgent need for larger and precise clinical trials.42 published a meta-analysis regarding the efficacy of MSC administration in lupus nephritis. The pooled results demonstrated that reduced ds-DNA, ANA, Scr, BUN, proteinuria, and renal sclerosis score were seen with MSC treatment. Lin et al.43 performed a meta-analysis to assess the efficacy of MSC treatment on diabetic kidney disease without species limitations. Their studies verified that MSC management can result in lower levels of BUN, Scr, and urinary protein, enhance the glycemic management and alleviate the renal fibrosis in animal DKD models. However, MSCs had minimal benefits in the DKD clinical trial. Lalu et al.44 conducted a meta-analysis regarding MSC therapy for stroke and confirmed that MSC therapy improves several neurological and motor function tests. However, poor effects of MSC therapy on stroke were observed in clinical studies. Moreover, according to the study of Wahid et al.45, MSC treatment made no difference in the 'no-option' critical lower limb ischemia patients. Obviously, there is a barrier in the bench to bedside translation of MSC therapy.In previous studies, MSCs showed an attractive potential in many other kidney and ischemic diseases. Zou et al.47. Hemodynamic changes often lead to tubulointerstitial hypoxia and activation of the RAAS system. Furthermore, tubulointerstitial hypoxia and the activation of the RAAS system can trigger oxidative stress and inflammation48, leading to tissue injury and interstitial fibrosis. Unlike acute hypoxia, chronic hypoxia resulted in microvascular remodeling and rarefaction, further accelerating the progression of renal fibrosis and dysfunction. Previous studies have shown that MSCs play an important role in angiogenic factors release, inflammation reduction, hemodynamic stabilization, and mitigation of oxidative stress, apoptosis, microvascular rarefaction, and fibrosis28. In the included preclinical studies, the suppression of the renin-angiotensin system in the MSC groups was confirmed by the decreased levels of PRA and the AT1 receptor and increased levels of the AT2 receptor, leading to the reduction of SBP. The microvascular regeneration detected by the micro-CT and the increased VEGF in the MSC groups might contribute to the increased RBF despite the unchanged patency of the renal artery. The increased GFR of STK seems to benefit from the increased RBF and renal fibrosis improvement. In the included clinical studies, although poor outcomes were described previously, Abumoawad’s and Saad’s studies discovered that RBF and perfusion were improved in the medical plus the MSC groups compared to baseline. Furthermore, Abumoawad et al. found dose–response changes in the estimated glomerular filtration rate, urine protein, and diastolic blood pressure. Saad et al. discovered dose–response changes in the RBF of STK and CLK. Kim’s study found that the efficacy of MSCs is decreased in SBP. However, the efficacy of reducing SBP was not significant when pooled with the data in Abumoawad’s studies. In addition, Abumoawad et al. and Saad et al. both indicated that inflammatory markers tended to improve. Nonetheless, the efficacy of MSCs in clinical trials is still doubtful.Our meta-analysis is the first research to evaluate the efficacy of MSC therapy in RAS. RAS gradually progresses over a long period of time. Complicated mechanisms including hemodynamic changes, tubulointerstitial hypoxia, and activation of the RAAS system are involved during the prolonged process37; thus, the frequency of MSC administration can also be taken into account. Fourth, the timing of MSC administration may also influence the efficacy of MSC delivery. In preclinical studies, MSCs were delivered from 2 to 6 weeks after renal artery clamping. MSC delivery was performed twice at the 3rd and 5th week within the four rat studies and once at the 6th week in all pig RAS models. MSC administrations were performed in the early stages of RAS in the preclinical studies. However, three clinical trials did not report the course of the disease. We speculated that there might be some enrolled patients in the chronic phase when the MSCs were delivered. Lerman et al.6 suggested that circulation of self-perpetuating tissue damage existed during RAS progression; therefore, the severity of RAS can be aggravated over time. Thus, the timing of MSC delivery as well as the course of RAS needs to be taken seriously.There are many possible reasons for the discrepancy of MSC efficacy between preclinical and clinical studies. First, the clinical trials were nonrandomized with relatively few patients and diabetic patients were excluded. Second, clinical trials were all at an early phase. The studies of Abumoawad et al. and Saad et al. are at phase 1a and phase 1/2a, respectively. The therapeutic conditions such as effective dosage of MSCs are still under-explored in the trials. Moreover, both studies ignored important clinical outcomes such as Scr, the degree of RAS and PRA. Kim et al. also performed a preliminary clinical study. Kim’s study provided deficient clinically relevant outcomes while they focused on the cellular senescence in STK. Third, dose–response changes should be evaluated. More significant changes and no adverse events were observed within the clinical outcomes when MSC doses was increased during the therapeutic intervention. Therefore, the dosage applied in the included trials might not meet the effective dosage. Proper dose escalation of MSCs should be considered in future trials. In addition, previous studies had verified that repeated MSC delivery brought benefits to rodent modelsAccording to the quality assessment results within the preclinical studies, none of the 16 articles met the ten criteria of low risk in the SYRCLE tool. Most studies only met three to five of the ten criteria. The major biases were the results of undefined random sequence generation methods, unclear allocation concealment, and the absence of the blinding of the animal breeders, researchers, and outcome assessors. The quality of the included clinical studies was at an intermediate level. The scores were deducted to exclude potential patients, lack of blinding, and calculation of the study size. Our quality assessment was limited by poor reports regarding the methodological protocols within the included articles. Thus, we sometimes had to make choices based upon our judgment. Therefore, future studies should show detailed protocols.The study has some limitations. First, the clinical studies were non-randomized controlled trials with few patients. High-quality RCTs and more comprehensive clinical outcomes are needed for a stronger conclusion. Second, the period after MSC intervention mostly ranged from 2 to 4 weeks within the preclinical studies and was set at 3 months in the clinical studies; thus, the long-term effects cannot be observed. Third, different types of MSCs and species were used, which influenced the conclusion of the preclinical studies. Fourth, all included studies did not report adverse events. However, the study sizes were limited; therefore, more extensive studies should be performed in order to verify the safety of MSC delivery. At last, due to the relative lack of related researches, this meta-analysis included both random and non-random studies without species limitations, which can lead to low quality of evidence. Abundant high-quality studies should be conducted to pool more reliable results in the future.In our meta-analysis, we provide preliminary evidences for the MSC therapy of RAS. In preclinical studies, MSC therapy might lead to decreased levels of SBP, Scr, PRA, IFN-γ, TNF-α, and MCP-1 and increased levels of RBF, GFR, microvascular density, and IL-10. Meanwhile, renal fibrosis can be alleviated after MSC therapy. In the clinical trials, only the cortical perfusion and fractional hypoxia of CLK benefits from MSC therapy. Nevertheless, this meta-analysis demonstrates that MSC therapy might be a potential therapy for RAS treatment. Although the MSC therapy isn’t recommended in clinical care right now, the cell-based therapy should attract more clinicians’ attention. In future, more well-designed basic studies should be performed to improve study quality, determine the optimal MSC delivery and dosage, and understand the global biological mechanisms of the observed protective effects. We hope that credible basic studies can provide important clues for the design of clinical trials and promote the bedside translation of MSC therapy.Supplementary Information 1.Supplementary Information 2.


The hippocampus is involved in various cognitive function, including memory. Hippocampal structural and functional abnormalities have been observed in patients with obstructive sleep apnoea (OSA), but the functional connectivity (FC) patterns among hippocampal subdivisions in OSA patients remain unclear. The purpose of this study was to investigate the changes in FC between hippocampal subdivisions and their relationship with neurocognitive function in male patients with OSA. Resting-state fMRI were obtained from 46 male patients with untreated severe OSA and 46 male good sleepers. The hippocampus was divided into anterior, middle, and posterior parts, and the differences in FC between hippocampal subdivisions and other brain regions were determined. Correlation analysis was used to explore the relationships between abnormal FC of hippocampal subdivisions and clinical characteristics in patients with OSA. Our results revealed increased FC in the OSA group between the left anterior hippocampus and left middle temporal gyrus; between the left middle hippocampus and the left inferior frontal gyrus, right anterior central gyrus, and left anterior central gyrus; between the left posterior hippocampus and right middle frontal gyrus; between the right middle hippocampus and left inferior frontal gyrus; and between the right posterior hippocampus and left middle frontal gyrus. These FC abnormalities predominantly manifested in the sensorimotor network, fronto-parietal network, and semantic/default mode network, which are closely related to the neurocognitive impairment observed in OSA patients. This study advances our understanding of the potential pathophysiological mechanism of neurocognitive dysfunction in OSA. Obstructive sleep apnoea (OSA) is one of the most common sleep disorders. It is characterized by the repeated collapse of the upper respiratory tract during sleep, resulting in occlusion or stenosis, sleep fragmentation, and a decrease in blood oxygen saturation. Among adults, approximately 14% of men and 5% of women are diagnosed with OSA. Obesity and increasing age are associated with an increased incidence of OSA . ChronicThe hippocampi of patients with OSA exhibit damage, which may contribute to the disordered memory, autonomic nervous system dysfunction, and depressive symptoms. Previous findings indicate that decreased hippocampal volume  and abnoUnderstanding the functional and structural impairments in the hippocampal subregions will provide insight into the neurocognitive mechanisms associated with OSA. Early rodent electrophysiological and recent gene expression studies showed that the dorsal and ventral parts of the hippocampus have separable response characteristics . These sTherefore, we hypothesized that FC abnormalities between the anterior and posterior axes of the hippocampus that participate in cognitive functions such as memory would be found in patients with OSA. Thus, we aimed to explore the FC between regions of interest (ROIs) in the anterior, middle, and posterior subdivisions of the hippocampus and the whole brain to assess the changes in FC patterns in patients with OSA. We also explored the relationships between the abnormal FC in these hippocampal subdivisions and clinical characteristics in patients with OSA.In this study, 46 untreated male patients with severe OSA diagnosed by polysomnography and an AHI of >30/h (OSA group) were recruited from the Department of Respiratory and Otorhinolaryngology of The First Affiliated Hospital of Nanchang University between September 2016 and June 2021. In addition, 46 good sleepers (GS group) of similar ages and education levels and with an AHI of <5/h were recruited from the community . The incDaytime sleepiness was assessed using the Epworth Sleepiness Scale (ESS) and neurocognitive function was assessed using the Montreal Cognitive Assessment (MoCA). The MoCA includes visual space and execution, naming, memory, attention, language, abstract thinking, calculation, and orientation . The higAll images were captured on a 3.0 Tesla MRI system with an eight-channel phased-array head coil . The subjects were instructed to lie quietly on the examination bed with their eyes closed, wore earplugs and goggles, had their head position stabilized with sponges, and were asked to try to clear their mind. First, conventional T1-weighted imaging was performed using the following parameters: repetition time (TR) = 4,000 ms, echo time (TE) = 113 ms, thickness = 5 mm, gap = 1.5 mm, field of view (FOV) = 220 mm × 220 mm, and slice thickness = 19. T2-weighted imaging was then performed using the following parameters: TR = 4,000 ms, TE = 113 ms, thickness = 5 mm, gap = 1.5 mm, FOV = 220 mm × 220 mm, and slice thickness = 19. Resting-state whole-brain blood oxygenation level dependent (BOLD) data were collected by echo-planar imaging using the following parameters: TR = 2,000 ms, TE = 30 ms, flip angle = 90°, FOV = 230 mm × 230 mm, matrix = 64 × 64, layer number = 30, slice thickness = 4.0 mm, interval = 1.2 mm, and scanning time of approximately 8 min. Finally, high-resolution three-dimensional T1-weighted images were obtained using the following brain volume sequence parameters: TR = 1,900 ms, TE = 2.26 ms, thickness = 1.0 mm, gap = 0.5 mm, FOV = 250 mm × 250 mm, matrix = 256 × 256, and flip angle = 9°, sagittal slices. After the MRI scan, all participants were asked if they had fallen asleep during the scan, and all images were examined by two senior radiologists to rule out significant brain damage.1 software package was used to evaluate the fMRI data, and data with incomplete image range, head movement, or large artifacts were removed. Next, based on the MATLAB 2012A  platform and the SPM 82 software package, the DPARSF V2.3 software package was used to pre-process the original data. The main steps were as follows: (1) The original DICOM format file was converted to NIFTI format. (2) The first ten time points were removed, and the remaining 230 volumes were corrected for time  and head motion  to eliminate physiological noise and potential head motion-related artifacts; these factors were compared between groups to assess their relative impact (3 × 3 mm3 × 3 mm3 voxels. (5) The image was then smoothed with a 6 mm half-width Gaussian smoothing kernel. (6) To further reduce confounding factors, nuisance covariates, including white matter signals, cerebrospinal fluid, whole-brain signals, and 24-parameter head movements were regressed from the time series of all voxels using linear regression  Date impact . (4) Difgression . (7) A tBased on previous guidelines, the hippocampus was anatomically divided into anterior, middle, and posterior regions. In this study, six ROIs were defined along the longitudinal axis of the left and right hippocampus with a radius of 3 mm . Each sit-tests were used to analyse normally distributed data and Mann–Whitney U-tests were used to analyse non-normally distributed data. For the analysis of ROI FC, the single sample t-test was first used to explore the spatial distribution pattern of FC in each hippocampal subregion and the whole brain in the two groups. For the analysis of the resting-state FC (rs-FC) of each ROI, two independent samples t-tests were applied to analyze intergroup differences with age, BMI, and years of education as covariates. Multiple comparisons were corrected using Gaussian Random-Field method . Based on the linear correlation analysis, the rs-FC value of each ROI was associated with clinical variables, and p < 0.05 was considered to be statistical significance.IBM SPSS 23.0 statistical software was used for the statistical analysis. The Kolmogorov–Smirnov test was performed to determine whether the data conformed to a normal distribution. To compare the differences in clinical evaluation between the OSA and GS groups, two-sample 2) of <90%, ESS score, MoCA score, and other variables  and positively correlated with orientation . The FC value between the left middle hippocampus and left inferior frontal gyrus was negatively correlated with N1  and positively correlated with N3 . The FC value between the left middle hippocampus and right precentral gyrus was negatively correlated with the MoCA scores , orientation , and total sleep time . The FC value between the left posterior hippocampus and right middle frontal gyrus was negatively correlated with total sleep time  and attention  and positively correlated with an SaO2 of <90% . The FC value between the left posterior hippocampus and left middle frontal gyrus was positively correlated with an SaO2 of <90% . Finally, the FC value between the right middle hippocampus and left inferior frontal gyrus was positively correlated with naming , which is involved in the regulation of negative self-referential thinking in major depression . The antThe anterior central gyrus belongs to the sensorimotor network and is the location of the primary motor cortex . Structu2 of <90%. Therefore, we speculate that this FC enhancement may serve as a potential compensation mechanism for the frontal parietal network affected by intermittent hypoxia injury.In addition, we found enhanced FC between the middle and posterior hippocampus and the middle frontal gyrus. The middle frontal gyrus belongs to the frontal-parietal control network, which is related to visuospatial planning, memory, attention, and cognitive control . Zhang eThe inferior frontal gyrus belongs to the semantic control network and is associated with controlled semantic retrieval . Other bThis study has some limitations. First, the sample size was small, which may restrict the representativeness of the results. Second, only patients with severe OSA were included, and those with mild and moderate OSA were excluded. Furthermore, this study did not include female patients, and the sex differences in the hippocampus may limit the generalizability of the results to the whole population. Future studies should use a larger sample size of patients with OSA and include female patients.We observed abnormal functional connections between the hippocampal subregions and the whole brain in male patients with severe OSA, and these abnormal functional connections mainly involved the sensorimotor network, semantic control network, and frontoparietal network, which are related to memory and cognition. These findings may be beneficial for investigating the potential neural mechanisms underlying cognitive impairment in patients with OSA.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University and therefore been performed in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki and its later amendments. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XL and LC wrote, reviewed, and revised the manuscript. DP guided and designed the MRI experiment. LC analyzed the resting-state fMRI data and results and wrote the manuscript. XL and WD analyzed and discussed the ideas of the manuscript. HL, LK, YS, PL, WX, and YZ collected resting-state fMRI data and applied for the ethics approval. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The subtilisin-like proteases (SBTs) are a large family of serine peptidases that are unique to plants. Previous studies have shown that SBTs are associated with developmental processes and environmental responses. However, comprehensive identification and systematic analysis of the SBT family have not been conducted in cotton. We used bioinformatics methods to analyze the structural characteristics, phylogenetic relationships, gene structures, expression modes, evolutionary relationships, selection pressures and stress responses of SBT gene family members in upland cotton. In this study, we identified 120 and 112 SBTs in the tetraploid cotton species G. hirsutum and G. barbadense, while 67 and 69 SBTs were identified in the diploid species G. arboreum and G. raimondii, respectively; these SBTs were divided into five distinct subfamilies. We identified the SBT gene GhSBT27A, and explore its function through virus-induced gene silencing and transmission electron microscopy. These results suggested that the GhSBT27A gene was involved in the response to drought stress. These results lay a foundation for further study on the drought stress mechanism of cotton. Serine proteases are an important class of protein hydrolases with serine as the active center. Almost one-third of the proteases in living organisms are serine proteases . SubtiliArabidopsis thaliana, with 56 SBTs, as well as some functions, have been identified  in accordance with theassays. The primers for the -ΔΔCt method was used to measure relative gene expression levels.manufacturer’s protocol. The experiments were independently repeated three times, and 2Gossypium hirsutum (ZJU), Gossypium barbadense (ZJU), Gossypium arboreum (CRI) and Gossypium raimondii (JGI), were obtained from the Cotton FGD  (https://cottonfgd.org/)  .https://pfam.xfam.org/) of peptidase S8 (PF00082), which most likely belongs to the subtilis protease gene family, was downloaded from Pfam. The protein sequence containing PF00082 was screened by HMMER software  (https://wolfpsort.hgc.jp/).The hidden Markov pattern (HMM) map  . The subTo investigate the evolutionary relationship among SBT genes, we performed multiple sequence alignments of the obtained genes using MEGA (MEGA7) and ClustalW . Based oArabidopsis thaliana and four cotton species , homologous sequences of subtilisins from these species were obtained by the procedure described above. Multiple sequence alignments were performed using MEGA7 and ClustalW software, and interspecies evolutionary trees were constructed by the maximum likelihood method.To explore the evolutionary relationship of SBT between the model crop Gossypium hirsutum using TBtools software , Gossypium barbadense (ZJU), Gossypium arboreum (CRI), and Gossypium raimondii (JGI), were obtained from the Cotton FGD  (https://cottonfgd.org/)  . MCScanXgd.org/) .https://www.ncbi.nlm.nih.gov/) , under different tissues and cold, heat, salt and PEG stresses from National Center for Biotechnology Information (ih.gov/) .http://:/bioinformatics.psb.ugent.be/webtools/PlantCARE/html/) was used to further analyze the cis-elements in the GhSBT gene promoter regions, and the cis-element information obtained was mapped using TBtools software   and synonymous substitutions (Ks) for duplicate genes .GhSBT27A) was silenced by virus-induced gene silencing. The VIGS vector was the laboratory storage vector pYL156, and we constructed pYL156:GhSBT27A with restriction enzyme cleavage sites XbaI and BamHI. The primers for the GhSBT-silencing fragment were as follows: forward primer, 5 ‘- aaggttaccgaattctctagaTTAATCAAAAGTTATAAAAGGAGCTTCA - 3’, reverse primer, 5 ‘- cgtgagctcggtaccggatccGGCTGCTGTGGATGCCGT - 3’. The VIGS (virus-mediated gene silencing) system consisted of the recombinant vector pYL156:GhSBT27A, positive control pYL156:PDS, negative control pYL156 and auxiliary vector pYL192. When plants reached the three-leaf stage, the control group was irrigated with aqueous solution, while the experimental group was irrigated with the same volume of 5% PEG6000.To study the function of the subtilis enzyme genes in the drought tolerance of cotton, one SBT gene (the highly expressed 3) were kept fresh to minimize mechanical damage such as pulling, bruising and crushing of the tissue and to minimize the sampling time. A Petri dish with electron microscope fixative was prepared before sampling, and the small tissue pieces were put into the Petri dish immediately after removal and cut into 1 mm3 pieces with a scalpel. The cut tissue blocks were then transferred to EP tubes with new electron microscope fixative for further fixation and pumped by vacuum until they sank to the bottom. Samples were left at room temperature for 2 h and then stored at 4°C. After postfixation, dehydration and permeation embedding, the samples were inserted into embedding plates and placed in an oven overnight at 37°C. The samples were washed three times with 0.1 M phosphate buffer PB (pH 7.4) for 15 min each time. We used Image-Pro Plus 6.0  as our transmission electron microscopy data analysis software.To investigate the effect of genes on the morphology of cotton cells, we used transmission electron microscopy to observe the morphological changes to chloroplasts in leaves of gene-silenced cotton plants. Inverted second leaves of the same size were selected for the control and treatment groups. The leaf tissues . Empty GFP vector without GhSBT27A gene was used as control.To observe the subcellular localization of GhSBT1A-GhSBT58A and GhSBT1D-GhSBT62D according to their position on the chromosome. Then, analysis of the physical and chemical properties of the amino acid sequence of cotton SBT gene family members was performed  was obtained from domain prediction and used to identify members of the SBT gene family in the whole cotton genome, as well as for comparison with the CDD, SMART, and other databases; genes with incomplete domains were removed manually. A total of 120 pairs of genes belonging to the SBT gene family were identified. The genes were renamed To analyze the evolutionary relationship between each member of the SBT family, ClustalW in MEGA7 software was used to compare 120 SBT protein sequences, and a rootless phylogenetic tree was constructed by the adjacency method (G. hirsutum (120), G. barbadense (112), G. arboreum (67), G. raimondii (69), and Arabidopsis (54) to construct an evolutionary tree , subclade SBT1 had the most members (153), and subclades SBT2, SBT4, and SBT5 contained 97, 81, and 49 genes, respectively. Notably, most of the SBT proteins in Arabidopsis and the four cotton species have corresponding homologous proteins in all subclades, suggesting that SBT proteins in these plants may be functionally conserved in dicotyledons. In addition, it has previously been shown that upland cotton and island cotton are the result of crosses between Gossypium arboretum and Gossypium raimondii. This was also confirmed by our finding that SBT proteins in tetraploid cotton (Gossypium hirsutum and Gossypium barbadense) and diploid cotton (Gossypium arboretum and Gossypium raimondii) always cluster together. In addition, the GhSBT and GbSBT pairs always cluster together, which also shows the importance of gene duplication during evolution.To better elucidate the phylogenetic relationship between the four cotton species and the Arabidopsis SBT genes, we used 422 protein sequences from Gossypium species. In Gossypium hirsutum, 119 genes were randomly distributed on 25 chromosomes, and 1 gene was localized on the scaffold. The number of SBT genes on each chromosome was between 1 and 11. Tandem replication occurred on chromosomes A06, A08, A10, and A12 and D06, D08, D10, and D12. There were 58 genes in the A subgenome and 62 genes in the D subgenome. There is no SBT gene on chromosome D01, which may be because these predicted SBT genes might have been duplicated or lost during evolution. In Gossypium barbadense, 112 genes were randomly distributed on 25 chromosomes, and there was no SBT gene on chromosome D01, similar to Gossypium hirsutum, which supports the gene duplication. The number of SBT genes on each chromosome was between 1 and 11. There were 54 genes in the A subgenome and 58 genes in the D subgenome. These results are similar to those for Gossypium hirsutum, indicating that the genetic evolutionary process of SBT genes is mature and stable. Tandem replication occurred on chromosomes A06, A10, and A12 and D05, D08, D10, and D12. In Gossypium arboretum, 64 genes were distributed on 13 chromosomes, and 3 genes were distributed on the scaffold, all of which were unevenly distributed. The number of SBT genes on each chromosome was between 1 and 13, there was at least 1 gene on chromosome A03 and up to 13 genes on chromosome A10, and tandem replication occurred on Chr05, Chr06, Chr08, Chr10, and Chr12 and 1 scaffold, tig00000498. In Gossypium raimondii, 68 genes were distributed on 12 chromosomes, and 1 gene was distributed on the scaffold, all of which were unevenly distributed. The number of SBT genes on each chromosome was between 1 and 12, there was no SBT gene on chromosome D02, and tandem replication occurred on Chr04, Chr08, Chr09, Chr10, and Chr11. In summary, both tandem and fragmental duplication are the main modes of gene amplification during the evolution of SBT genes.To further study the chromosomal distribution and gene replication of SBT genes in four Gossypium species, we mapped the physical locations of these genes on chromosomes Figure 2GhSBT family members, phylogenetic trees, motif association analysis and gene structure analysis of GhSBT genes were performed  in different tissues  and generated a heatmap , we visualized the relationships between SBT genes from the four cotton varieties  and synonymous substitution rate (Ks) of two protein-coding genes was calculated to determine whether there was selective pressure acting on them. Synonymous mutations are not thought to be subject to natural selection because they do not affect amino acid sequences or protein structure or function. Nonsynonymous mutations, on the other hand, are subject to natural selection because they can affect amino acid sequences and may alter protien structure and function. Therefore, if Ka/Ks > 1, it is considered that there is positive selection effect. If Ka/Ks = 1, neutral selection is considered to occur. If Ka/Ks < 1, a negative selection effect is considered to be present, i.e., a purification effect or purifying selection.The Ka/Ks values of 964 gene pairs in 4 cotton species were calculated by TBtools software Figure 6GhSBT27A, from our PEG stress transcriptome data for further study. To investigate the effect of GhSBT27A on PEG stress, we detected the relative expression levels of GhSBT27A in the roots, stems and leaves under PEG stress, indicating that the gene was successfully knocked down. The above cotton plants were treated with a 5% concentration PEG. After 6 h of treatment, pYL156:GhSBT27A plants exhibited a more severe wilting phenotype, while control plants (PEG-pYL156) were relatively less affected  genome, 120 SBT genes were identified by BLASTP. These genes encode proteins with 136-2,111 amino acids, a molecular weight of 15.289-226.819 kDa, and an isoelectric point of 4.387 to 10.369, with an average of 7.382, suggesting that these proteins are weakly basic. The members of the SBT family encode proteins with different physicochemical properties and different functions and regulatory mechanisms, but they all share a stable SBT structural domain . The resGossypium hirsutum, Gossypium barbadense, Gossypium arboreum, Gossypium raimondii, and Arabidopsis thaliana. An evolutionary tree was constructed based on their evolutionary relationships. The evolutionary tree divided these genes into five branches, each of which contained genes for SBTs present in all five species. It was shown that SBT genes are present not only in cotton and Arabidopsis but also in other monocotyledonous species, indicating that SBT genes are present in the genomes of both dicots and monocots. This means that the SBT gene family formed subgroups I, II, III, IV and V before the separation of dicots and monocots, and it has also undergone different periods of differentiation. Analysis of the gene structures and conserved motifs of the SBT family members showed that these five branches have similar gene structures and conserved motifs (GhSBT26 and GhSBT27A are tandemly repeated genes. Tandem, segmental and whole genome replication play a crucial role in the expansion of gene families.In this study, we identified and analyzed the SBT family genes of five species, including d motifs . The pred motifs . Among tIn terms of chromosomal location, SBT genes were unevenly distributed in the At and Dt subgroups. However, the distribution patterns converged in both subgenomes, with both having SBT genes present on most chromosomes. Most of the SBT genes were distributed at both ends of the chromosome, while a few genes were distributed in the middle. The stable heritability of these genes indicates the importance and wide range of functions for the SBT gene family .We calculated the Ka/Ks ratio of 964 gene pairs to investigate the influence of selective pressure on SBT genes during evolution. Among them, there were 942 duplicate gene pairs with Ka/Ks < 1, indicating purifying selection. There were 22 genes with Ka/Ks > 1, which indicated that these genes were rapidly evolving in recent years and had very important significance for the evolution of the species. In the cotton SBT family, 97.72% of the gene pairs underwent purifying selection, indicating that strong purifying selection occurred after tandem duplication, fragment duplication and whole genome duplication in this gene family. However, the selection pressure of most gene pairs was between 0 and 0.49, indicating that SBT gene pairs tended to be conserved during evolution .GhSBTs genes were associated with various stresses, such as drought, low temperature, and defense, as well as hormone responses, such as responses to abscisic acid, gibberellin, salicylic acid, growth hormone, and MeJA. This result indicates that GhSBTs are not only involved in multiple signaling pathways but also in plant growth and development and defense responses, providing a reference for screening for stress resistance genes (GhSBT27A gene contains multiple abscisic acid response elements, which are consistent with our findings that this gene regulates drought resistance in upland cotton.When plants are subjected to abiotic stresses, cis-acting elements upstream of each gene play an important role. These cis-acting elements do not encode proteins, but they can regulate gene expression . The cisce genes . The GhSGhSBT27A may affect plant drought tolerance by affecting chloroplast structure. Our data showed that under PEG stress, the relative expression levels of GhSBT27A were up-regulated, the relative expression level of GhSBT27A in roots under PEG is the highest. We used virus-induced gene silencing to silence GhSBT27A, and under PEG stress, GhSBT27A-silenced plants were more severely stressed than the control, suggesting that the GhSBT27A gene plays an important role in drought stress resistance in cotton. Moreover, transmission electron microscopy results showed that chloroplast degradation was more severe in GhSBT27A-silenced plants than in control plants, which further suggested that the GhSBT27A gene might resist drought stress by regulating chloroplast morphological structure.Based on the results of our study, we hypothesized that GhSBTs in upland cotton, which underwent tandem and genome-wide duplication during evolution. GhSBTs were classified into five branches based on the phylogenetic tree, gene structure and motif analyses. The study of GhSBT27A, a member of evolutionary branch 1, revealed that it plays an important role in regulating drought stress in cotton. This study enriches the understanding of upland cotton SBT genes and lays the foundation for further studies on the functions of GhSBTs in cotton.This study provides the first comprehensive analysis of the SBT gene family in cotton. Here, 368 SBT genes were detected in four cotton species, including 120 https://cottonfgd.org/, https://pfam.xfam.org/.Publicly available datasets were analyzed in this study. This data can be found here: QC designed the project. MD, NZ and YZ conducted the experiments. MD wrote the manuscript. NZ and YXZ performed the bioinformatics analysis. NZ, YXZ, KN, ZW, LL and XW assisted in writing and editing. QC was responsible for revising the manuscript. All authors have read and approved the final manuscript. All authors contributed to the article and approved the submitted version.


The combination of chemotherapy, targeted therapy, and immunotherapy provides an effective antitumor regimen for advanced NPC patients with BMI.Bone marrow involvement (BMI) of solid tumors is a special type of distant metastasis. It has an occult onset, atypical clinical and laboratory features, and a high mortality. We present a nasopharyngeal carcinoma case with cervical and axillary lymph nodes, bilateral lung, multiple bone, and bone marrow metastases, who was treated chemotherapy plus targeted therapy under the guidance of a patient-derived tumor xenograft (PDTX) model, followed by maintenance chemotherapy plus immunotherapy. The patient’s symptoms were relieved after four cycles of chemotherapy plus targeted therapy. His bone marrow biopsy turned negative after 7 months of therapy. In addition, his total peripheral T cells as well as the proportion of CD8 Bone marrow involvement (BMI) in solid tumors is rare in clinical practice compared with that of hematological tumors. The incidence rate of BMI in solid tumors is reported to range from 11% to 20% , which iThe incidence rate of BMI in nasopharyngeal carcinoma (NPC) is unclear. Berry reported a 63-year-old man with NPC and BMI who died within 2 weeks of the diagnosis without antitumor therapy . Miyaush2, every 3 weeks) plus nedaplatin  were conducted from January 4, 2020, to March 4, 2020. Partial remission was achieved after induction chemotherapy, assessed by the nasopharyngeal magnetic resonance imaging (MRI) and cervical CT scan, according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Intensity-modulated radiotherapy (70 Gy/32 fractions) with two cycles of nedaplatin  concurrent chemotherapy and fixed-dose weekly nituzumab (200 mg) concurrent anti-EGFR-targeted therapy was conducted from March 13, 2020, to April 28, 2020. The patient received one cycle of adjuvant chemotherapy of paclitaxel-albumin plus nedaplatin. Complete remission was achieved in the imaging evaluation 1 month after the completion of radiotherapy. From then on, he had regular visits until August 2020, when no recurrence and metastasis were detected.A 44-year-old man was initially diagnosed in our center with stage IVa  8th edition) NPC and dermatomyositis in December 2019. Positron emission tomography–computed tomography (PET-CT) showed the primary tumor was confined to the nasopharynx, accompanied by lymph node metastasis in the left neck, left parapharyngeal space, and left clavicle. The diagnostic and treatment history of the patient is shown in The patient suffered from lumbar pain on October 9, 2020. MRI and PET-CT scans revealed right scapula, right 3rd rib, left 6th rib, thoracic 10 to lumbar 5 vertebral bodies and some accessories, sacrum, bilateral iliac bones, right ischium, left upper femur metastases, and lung metastases. Subsequently, he underwent posterior thoracolumbar tumor resection, reconstruction, and internal fixation under general anesthesia on October 27, 2020. The postoperative pathology was metastatic nonkeratinized undifferentiated carcinoma, which was confirmed as a bone metastasis of NPC. PDTX models were built using cancer cells obtained from surgery to evaluate the sensitivity of different regimens of systemic therapy.9/L , glutamic-pyruvic transaminase (GPT) of 85 U/L , glutamic oxaloacetic transaminase (GOT) of 99 U/L , alkaline phosphatase (ALP) of 243 U/L , lactate dehydrogenase (LDH) of 1,085 U/L , and C-reactive protein (CRP) of 134.9 mg/L . He had no liver metastasis or chronic liver disease, and dermatomyositis was controlled in a stable state. Based on his general situation and the result of the PDTX model, he was administered gemcitabine  plus cetuximab  from November 29, 2020. After two cycles of treatment, the patient’s temperature returned to normal (below 37.4°C), the pain eased, the KPS score increased to 80, and partial remission was found in all lymph nodes, lung, and bone lesions at the first assessment after two cycles of treatment. Cisplatin  was added to the regimen beginning with the 3rd cycle of therapy. Repeated MRI and CT scans showed continued partial remission after four and six cycles of treatment. ALP and LDH levels continuously declined. The dynamic changes of ALP and LDH over the treatment are exhibited in He was readmitted on November 17, 2020. At that time, he had a Karnofsky performance status (KPS) score of 50, a continued fever of more than 39°C, and lumbar pain, which caused him to be bedridden for 3 weeks after the operation. MRI and PET-CT scans revealed that he had rapid progression with bone and lung metastasis. BMI was detected from bone marrow aspiration and biopsy  for the publication of any potentially identifiable images or data included in this article.QW and BZ contributed to the study concept and design. TZ contributed to the investigation and writing of the original draft. LJ contributed to the collection of biopsy data and analysis. YZ contributed to the collection of CT image data and analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.