| Background and objectiveCraniopharyngioma(CP)is a benign intracranial tumor including two histological subtypes,i.e.,adamantinomatous craniopharyngioma(ACP)and papillary craniopharyngioma(PCP).ACP as the main subtype of CP,accounts for nearly 90%of CP cases.The GTR of tumor is still the mainline treatment modality for CPs.However,although CP is a biological benign and curable tumor,the close relationship between tumors and the hypothalamus-pituitary axis(HPA)makes the incidence of postoperative hypothalamic damage up to 68%for CP patients.As is well known,hypothalamic obesity(HO)is the key factor associated with the poor quality of life and less survival time for CP patients.Yet evidence is mounting that hypothalamic damage is significantly associated with postoperative HO in childhood CP patients.However,this association need to be further confirmed in adult-onset CP patients to highlight that the prevention or reduction of hypothalamic damage is pivotal for the management of CP patients.As one of the characteristic pathological structures of ACP,finger-like protrusion is frequently found in the tumor specimens contacting with the surrounding hypothalamic tissue,showing that the tumor extends into the surrounding hypothalamic tissue with finger-like protrusion.Of note,it is an important cause of postoperative hypothalamic injury.However,the formation mechanism of finger-like protrusion of ACP is not clear,and effective clinical intervention for finger-like protrusion is missing.Our previous studies and related reports have shown that the tumor microenvironment in the brain tissue surrounding ACP and whorl-like cell clusters inside the tumor may play an important role in the formation of finger-like protrusion.This study aims to first reveal whether the hypothalamic damage is an independent risk factor for HO in adult-onset CP patients,aiming to highlight that the prevention and reduction of hypothalamic damage is pivotal for decreasing the rate of HO and improving the prognosis for CP patients.Furthermore,by integrating single-nucleus sequencing(sn RNA-seq)and spatial transcriptome(ST),the potential formation mechanism of ACP finger-like protrusion leading to hypothalamic damage,was explored through focusing on the tumor microenvironment of surrounding hypothalamic tissue and the whorl-like cell clusters inside ACPs,aiming to provide potential drug targets for clinical intervention in the formation of finger-like protrusion,thereby reducing or even eliminating hypothalamic injury during the GTR of tumor and ultimately improving the prognosis of ACP patients.Materials and Methods1.The data of surgically treated patients with CP at our center from December2009 to October 2021 were retrospectively reviewed.Patients with age≥18years,complete medical data,and primary CPs were enrolled.The status of hypothalamic damage was evaluated according to the Puget’grading system,Muller’grading system,Roth’s hypothalamic lesion score and the hypothalamic injury pattern proposed by our team,which were based on postoperative magnetic resonance images(MRIs)and intraoperative endoscopic observation,respectively.The main outcome measures in this part of the study were clinically meaningful weight gain(≥5%)and HO(body mass index[BMI]≥28.0 kg/m~2and increase in BMI≥2 kg/m~2 within 1 year before surgery or 6 months after surgery).Firstly,the changes of body weight,BMI and obesity rate at different points in time(before surgery,6 months after surgery,1 year after surgery and the last follow-up)were depicted.Then,univariate and multivariate logistic regression analysis was used to determine whether hypothalamic damage evaluated by the abovementioned grading systems was an independent risk factor for postoperative weight gain and HO.2.Three frozen ACPs with finger-like protrusion into the hypothalamus,which was confirmed by histologic examination were collected.To characterize intra-tumoral heterogeneity in a whole tumor,two samples,one close to the hypothalamus and one distant from the hypothalamus,were obtained from each tumor.Additionally,to provide spatial information for the sn RNA-seq data,a total of four ST sections were obtained,two from one patient for sn RNA-seq and two from two other patients with pathologically confirmed ACP;similar to the samples undergoing sn RNA-seq,two ST sections were excised close to the hypothalamus and the others were distant from the hypothalamus.Firstly,we unraveled the cellular component of ACPs with finger-like protrusion into the hypothalamus via sn RNA-seq.Then we extracted epithelial cell and neuron to further depict their cellular heterogeneity by integrating sn RNA-seq and ST.Finally,the interplay between epithelial cell and neuron was explored using cell-cell communication analysis.3.In this part,we explored the potential mechanism of the whorl-like cell clusters driving the formation of ACP finger-like protrusion.We first collected 35 ACP samples that invaded the hypothalamus with finger-like protrusion.Two specimens for each tumor that one is from the fusion site of the tumor and hypothalamus and the other is from the area with a clear interface between the tumor and hypothalamus,were collected for histological examination.The differences in pathological structure between these two specimens were identified.Moreover,the correlation between the depth of finger-like protrusions and the size of whorl-like cell clusters was determined by correlation analysis.Then,cells with nuclear/cytoplasmicβ-catenin accumulation identified in the previous part of this study were extracted.After correcting the batches and re-clustering,the subtypes of cells with nuclear/cytoplasmicβ-catenin accumulation were identified.By integrating ST,immunofluorescence(IF)and fluorescence in situ hybridization(FISH),the spatial distribution of each subgroup and the cell composition of whorl-like clusters in finger-like protrusion were determined.Further,the Kyoto Encyclopedia of Genes and Genomes(KEGG)and hallmark pathway enrichment analysis of each subpopulation was conducted using Gene set variation analysis(GSVA),results of which were verified by IF.Results:1.In this part of study,forty-seven(49.0%)patients and 18(18.8%)patients experienced clinically meaningful weight gain(≥5%)and HO at last follow-up,respectively.Postoperative weight significantly increased during the first 6 months following surgery,followed by stabilization.Both grade 2 postoperative hypothalamus damage,as evaluated by the MRI classification system proposed by Müller et al.,and higher scores based on the Roth et al.hypothalamic lesion score were significantly associated with postoperative weight gain of≥5%(P=0.005 and P=0.002)and with HO(P=0.001 and P=0.008).Additionally,hypothalamic injury as evaluated by the hypothalamic injury pattern based on endoscopic observation proposed by our team(P=0.008)could predict postoperative weight gain≥5%.2.In this part of study,we first revealed the cell types that constitute ACP tissues from patients with tumor finger-like protrusion into the hypothalamus,including epithelial cells,B cells,plasma cells,dendritic cells(DCs),endothelial cells,fibroblasts,macrophages,monocytes,natural killer(NK)&T cells,astrocytes,neurons and oligodendrocytes.Furthermore,for the first time,we comprehensively revealed epithelial cell heterogeneity in ACP,showing that EC1 has the largest number of cells and represents the majority of tumor cells,EC2 represents cells undergoing calcification/mineralization,EC3 subtype represents cells with nuclear/cytoplasmicβ-catenin accumulation,EC4 harbors neuronal attributes,EC5 has the highest proliferation potential,and EC6 may involve in antimicrobial immune response.Additionally,we intensively explored the molecular features of neurons in ACP tissues,and a group of newborn and inhibitory neurons was discovered,while it was lack in ACP tissues without finger-like protrusion into the hypothalamus.Further,we compared the expression of GABA,ABAT,GAD67 and GAD65 between ACPs invading hypothalamus with finger-like protrusion and ACPs without finger-like protrusion into the hypothalamus by immunohistochemistry(IHC),which showed that GABA and ABAT significantly increased in ACPs invading the hypothalamus with finger-like protrusion and no significant difference in GAD67 and GAD65 between the these two groups,suggesting that GABA metabolism is up-regulated in ACPs with finger-like protrusion into the hypothalamus and GABA is derived from surrounding hypothalamic tissues.Moreover,via a cell?cell communication analysis,we found that NGR1/NGR2/NGR3-ERBB4 were likely the most active ligand?receptor pairs between EC4 cells and neurons.To support these potential interactions,we mapped the expression of the ligand?receptor pairs to one ACP tumor with two ST slides,one close to the hypothalamus and one distant from the hypothalamus.Notably,NRG1/NRG2-ERBB4 coexpression spots in the borders of the hypothalamus and invading ACP tumors were extensively observed and significantly increased in ACP ST slices invading the hypothalamus with finger-like protrusion.3.In this part,we found that in each ACP involving the hypothalamus,whorl-like cell clusters were present in all finger-like protrusions and were absent in the specimens in contact with the hypothalamus without finger-like protrusion,but there were no significant differences in the stellate reticulum and peripheral pallisading epithelium between them.In addition,the depth of finger-like protrusions was positively correlated with the size of whorl-like cell clusters,suggesting that the expansive growth of whorl-like cell clusters may drive the formation of ACP finger-like protrusion.Further,we revealed that cells with nuclear/cytoplasmicβ-catenin accumulation can be divided into three subgroups with different molecular characteristics,i.e.,GRM8~+,CD133~+and RSPO2~+subtypes.Whorl-like cluster consisted of GRM8~+and CD133~+cells.Moreover,GRM8~+and CD133~+cells were co-located with cells with nuclearβ-catenin accumulation and cytoplasmicβ-catenin accumulation,respectively,and they were adjacent to each other,while RSPO2~+cells were mainly located around wet keratin/ghost cells.CD133~+cells are hyperproliferative,where the IL-6-JAK2-STAT3signaling pathway is activated.Meanwhile,GRM8~+cells had senescence-associated secretory phenotypes.Conclusions:1.Hypothalamic damage is an independent risk factor associated with postoperative HO in adult-onset CP patients.However,HO is refractory to current treatments,making the prevention and reduction of hypothalamic damage pivotal for the treatment of patients with CPs.2.ACPs invading the hypothalamus with finger-like protrusion consist of diverse cell types.Epithelial cells in ACPs showed significant heterogeneity,including 6epithelial subtypes with different molecular functions.Neurons in the hypothalamus tissue adjacent to ACP are mainly a group of newborn neurons with inhibitory neuron properties.In addition,epithelial cell-neuron interactions may play an important role in ACP invading the hypothalamus with finger-like protrusion.3.By integrating sn RNA-seq,ST and IF,we tentatively suggested that GRM8~+cells in whorl-like cell clusters may secrete IL-6,and the latter acts on CD133~+cells through paracrine mode to activate JAK2-STAT3 signaling pathway,thus promoting their proliferation and further leading to the expansive growth of whorl-like cell clusters,which eventually drives the formation of ACP finger-like protrusion. |
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