following four washes in pbs, sections were incubated for 90 s with trueview ( vector laboratories ), washed by immersing in pbs for 5 min, then tap - dried and mounted in vectashield vibrance™ antifade. sections were imaged using a zeiss lsm 710 confocal microscope at ×20 ( plan - apochromat ×20 / 0. 8 m27 ) and ×63 ( plan - apochromat ×63 / 1. 40 oil dic m27 ) magnification. tile scans were set to cover an area of 3541 × 3542 microns for all sections. imagej was used to remove background bodipy signals and calculate the area covered by the thresholded bodipy on the stitched images. to compare the area covered by bodipy in tumour and background, we used a paired t test at a patient level, after confirming the normal distribution of the data using a shapiro – wilk test. to investigate the oncofetal reprogramming of myeloid cells in nsclc, we took advantage of a published scrna - seq dataset of foetal lung myeloid cells and the published “ momac - verse ”. the expression of the “ stab1 signature genes ” and of the “ amɸ signature genes ” across lung foetal myeloid cells was determined using the addmodulescore function in seurat v4. 3. to combine foetal lung and adult lung tumour - infiltrating myeloid cells, we isolated the myeloid cells from our tumour and background datasets and integrated those with the aforementioned foetal lung myeloid dataset using the pegasus package, following the following workflow: ( i ) remove rarely expressed genes ( less than 10 cells ), normalisation and log1p transformation, ( ii ) robust and highly - variable gene selection, ( iii ) pca with optimal pc number determined by random matrix theory ( resulting in 75 pcs ), ( iv ) batch effect correction using harmony, and ( v ) leiden clustering on neighbourhood graph. the dendrogram was built by estimating the correlation distance between cell types on the harmonised pc embedding space, under complete linkage criterion of hierarchical clustering. the umap was computed to obtain a 2d summary of the harmonised pc space. tissues were frozen in dry - ice - cooled isopentane and stored in air - tight tissue cryovials at −80 °c. prior to undertaking any spatial transcriptomics protocol, the tissues were embedded in oct compound and tested for rna quality with an agilent bioanalyser. tissues with rna integrity ( rin ) values > 7 were cryosectioned in a pre - cooled cryostat at 10 μm thickness. two consecutive sections were cryosectioned at 10 μm thickness in a pre - cooled cryostat and transferred to the four 6. 5 mm × 6. 5 mm capture areas of the gene expression slide. slides were fixed in methanol for 30 min prior to staining with h & e and then imaged using the nanozoomer slide scanner. the tissues underwent permeabilization for 24 min. reverse transcription and second strand synthesis was performed on the slide with cdna quantification using qrt - pcr using kapa sybr fast - qpcr kit ( kapa biosystems ) and analysed on the quantstudio ( thermofisher ). following library construction, these were quantified and pooled at 2. 25 nm concentration. pooled libraries from each slide were sequenced on novaseq sp ( illumina ) using 150 base pair paired - end dual - indexed set - up to obtain a sequencing depth of ~ 50, 000 reads as per 10x genomics recommendations. the sequencing libraries were then processed by spaceranger ( version 1. 1. 0 ) on the reference grch38 human reference genome to estimate gene expression on spots. we used cell2location to deconvolute the cellular composition of each capture area ( spot ). as our scrna - seq cells were annotate d independently for tumour and the combined b / h datasets, we applied the deconvolution model separately as well, using tumour annotation to infer spatial cell composition of tumour sections, and background annotations for background datasets. only spots with total umi counts above 800 were used in downstream analysis. the cell - type abundance in tumour and background sections were computed by summing up the q05 cell abundance, as estimated by cell2location, across spots that passed qc. cell - type composition was computed by normalising each cell type ’ s abundance with the total abundance of all cell types. we compared cell - type composition between tumour and background with wilcoxon signed - rank test, followed by bonferroni correction. on tumour sections, we estimated the correlation distance on cell - type composition across valid spots, applied hierarchical clustering with complete linkage, and visualised the results as a dendrogram. in addition, we applied non - negative matrix factorisation analysis to the q05 estimation of cell - type abundance with eight factors. to study the expression of ligand – receptor pairs on the 10x visium, we first binarised the expression of each gene in the lr pairs in the spots that passed qc. we considered a gene being expressed in a spot if its cell2location estimated abundance were higher than the median counts for that gene in the corresponding section. we counted spots where both genes in each lr pair were either co - expressed or not, in tumour and background sections from the same patient, and subsequently, applied the χ test on the contingency table. to correct for multiple comparisons, we adjusted the p value using a conservative bonferroni correction for all the lrs enriched in tumours in the cellphonedb analysis ( 309 * 8 patients ). lrs were considered significantly enriched in tumour if the bonferroni - adjusted p value was lower than 0. 05 in at least four patients. 5 μm thick sections were generated from nsclc ffpe tumour blocks. an antibod y cocktail was prepared with optimal dilutions of each of the following conjugated antibod ies: anti-human stabilin - 1 antibod y ( clone # 840449, catalogue # mab3825, r & d systems ) was conjugated to a custom oligo barcode according to instructions in akoya biosciences ’ antibod y conjugation kit ( conjugation kit, # 7000009; akoya ) while human cd68 ( clone # kp1, catalogue # 4550113, akoya ) and human panck ( clone ae - 1 / ae - 3, catalogue # 4150020, akoya ) were obtained directly pre - conjugated to oligo barcodes from akoya biosciences. complementary oligo - conjugated fluorophore reporters were obtained from akoya biosciences. tissue multiplexed immunoflu orescence staining and image acquisition were performed according to akoya phenocycler - fusion user guide ( pd - 000011 rev. a., akoya ). ome - tiff files were generated and processed for image analysis. analysis of the multiplexed immunoflu orescence images ( generated from akoya phenocycler - fusion platform ) was performed using visiopharm ( version 2023. 09. 3. 15043 × 64 ) on the entire tissue area. briefly, cell segmentation ( including both nuclear and cytoplasmic segmentation ) was first performed using visiopharm ’ s “ cell detection, ai ( fluorescence ) ” ( version 2023. 09. 3. 15043 × 64 ) with its default parameters. after cell segmentation, visiopharm ’ s “ phenoplex guided workflow ” was used. dapi ( nucleus ), cd68 ( cell body ) and stab1 ( cell body ) variables were selected and manually thresholded to define positive and negative cells for each marker and generate a co - occurrence matrix. macrophages were defined as [ dapi +, cd68 + ] while stab1 + macrophages were defined as [ dapi +, cd68 +, stab1 + ]. further information on research design is available in the nature portfol io reporting summary linked to this article. bi - allelic variants in popdc2 cause an autosomal recessive syndrome presenting with cardiac conduction defects and hypertrophic cardiomyopathy source paper: pmc12256823 popdc2 encodes the popeye domain - containing protein 2, which has an important role in cardiac pacemaking and conduction, due in part to its cyclic amp ( camp ) - dependent binding and regulation of trek - 1 potassium channels.