Biology subjects

Sivakumar, S.

Publications and source records attributed to Sivakumar, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic CRISPRi perturbation of 1,408 autism risk genes maps multilevel transcriptional convergence in human cortical neurons

Genetic heterogeneity in autism spectrum disorder (ASD) complicates the identification of shared molecular pathways amenable to therapeutic intervention. Here, we perform a CRISPRi Perturb-seq screen targeting 1,408 ASD risk genes in human embryonic stem cell-derived immature cortical neurons and profile the resulting transcriptomic effects by single-cell RNA sequencing. We identify 215 ASD risk genes whose repression induces significant global transcriptomic dysregulation. Leveraging this functional atlas, we characterize candidate genes based on transcriptional similarity to established ASD hub genes. We identify gene programs recurrently dysregulated across perturbations, anchored by processes governing microtubule dynamics, neuron differentiation, cell migration, and transmembrane transport. We further identify CHAMP1 as a previously unrecognized regulator of Wnt signaling and define specific ASD risk genes that modulate the rate of cortical neuron differentiation. Analysis of differentially expressed genes reveals both convergent and perturbation-specific downstream transcriptional responses. Together, these findings provide a multilevel map of transcriptomic convergence in ASD and establish a framework for identifying both pathway-level and genotype-specific therapeutic strategies.

neuroscience↗

bioRxiv2026

Breast cancer ovarian metastases show increased activity of GPCR pathways

Treatment resistance and metastases occur in 10-20% of patients with invasive lobular carcinoma (ILC), the most common special histological subtype of breast cancer. ILC metastasizes to the ovary more frequently than no special type (NST) tumors, also known as invasive ductal carcinoma (IDC). To characterize the genomic landscape of breast cancer ovarian metastases, we analyzed 15,613 local breast cancers, 22,010 non-ovarian metastases, and 246 ovarian metastases sequenced using FoundationOne(R)CDx or FoundationOne(R) assays. Ovarian metastases had enriched CDH1, PIK3CA, and TBX3 mutations and depleted TP53 and MYC alterations relative to local breast cancers, with additional depletion of ESR1 mutations compared to non-ovarian metastases. CDH1 mutations were less frequent in ovarian metastases (47%) than local ILC (81.3%), with reduced 16q loss (64% vs 84%), indicating that ovarian metastases also arise from non-ILC tumors. We extended these findings to a UPMC cohort of 27 ovarian metastases (13 ILC, 8 IDC, 6 mixed ductal-lobular carcinoma) with patient-matched primary tumors in most cases. In both cohorts, patients with ovarian metastases were significantly younger than those with other metastatic sites. In the UPMC cohort, the most frequent mutations were in PIK3CA, CDH1, KMT2C, FOXA1, and RUNX1. Transcriptomic analysis identified upregulated G protein-coupled receptor (GPCR) pathways, including metabotropic glutamate receptor signaling. Functional studies showed that calcium-sensing receptor (CaSR), a GPCR overexpressed in ovarian metastases, drives MEK/ERK-dependent migration and F-actin reorganization in ILC cell lines, enhanced by estrogen and blocked by calcilytic, MEK, or anti- estrogen treatment. Our findings inform future therapeutic targeting of ovarian metastasis.

cancer biology↗

bioRxiv2026

Dietary omega-6 arachidonic acid and omega-3 docosahexaenoic acid supplementation differentially impact skeletal muscle inflammaging in mice

Aging is associated with a gradual and progressive decline in skeletal muscle mass and strength known as sarcopenia, which has been attributed to chronic low-grade inflammation. Dietary long-chain polyunsaturated fatty acids (LC-PUFAs), including omega-6 arachidonic acid (ARA) and omega-3 docosahexaenoic acid (DHA), are precursors to bioactive lipid mediators that regulate the initiation, propagation, and active resolution of inflammation. While traditionally considered a pro-inflammatory and catabolic factor, the ARA-derived eicosanoid prostaglandin E2 has recently emerged as a potential anti-sarcopenic molecule. DHA-derived specialized pro-resolving mediators may also act as immunomodulatory pro-regenerative molecules in muscle inflammaging. In the current study, we tested the effects of long-term dietary supplementation with either ARA or DHA on muscle health in aging mice. Twenty-two-month-old C57BL/6N mice were fed a control AIN-93M diet, or an AIN-93M diet supplemented with either ARA (0.48% w/w) or DHA (0.48% w/w) for 12 weeks. Both dietary interventions reduced total body weight, but only ARA reduced absolute fat mass and increased the percentage of lean mass. Despite these changes in body composition, ARA supplementation reduced absolute muscle strength and myofiber size. This functional decline was associated with increased neuromuscular junction fragmentation, elevated expression of pro-inflammatory cytokines/protein degradation markers, and suppressed ribosome biogenesis. In contrast, DHA uniquely reduced chronic inflammation of aged muscle and returned c-Myc expression to young levels but did not affect muscle mass or strength. These data demonstrate that long-term dietary intake of ARA and DHA have overall divergent effects on the structure and function of aging muscle.

physiology↗

bioRxiv2026

Landscape of reversion alterations in homologous recombination genes reveals evolutionary constraints

Reversion mutations (REVs) restore homologous recombination repair (HRR) and confer resistance to PARP inhibitors (PARPi) in HR-deficient cancers. Yet, their prevalence, mechanisms, and biological constraints remain undefined. We analyzed genomic profiling of 609,464 tissue and liquid biopsy samples across multiple cancer types to delineate the pan-cancer landscape of REVs. REVs were identified in eight HRR genes, most frequently BRCA2 and BRCA1 and notably never in ATM or CHEK2. REVs exclusively impacted truncating pathogenic variants, predominantly through large in-frame and exon-level deletions, associated with repetitive sequences. Conserved functional domains are relatively depleted of REVs. Structural modeling and functional studies support that exon-level deletions preserve critical domain architecture and confer PARPi resistance. The study establishes HRR reversion as a structurally permissive, yet evolutionarily constrained, resistance mechanism with implications on response, monitoring, and therapeutic strategy. One Sentence SummaryHomologous recombination reversion evolves through structurally tolerated, microhomology-driven alterations that restore DNA repair under therapeutic selection across cancers.

cancer biology↗

bioRxiv2026

Coordinated immune architecture underlies durable survival in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterised by profound immune dysfunction and limited response to immunotherapy. Although tertiary lymphoid structures (TLSs) are associated with improved outcomes, most studies focus on their presence or density, providing limited insight into how their organisation contributes to tumour control. Here, we analyse a unique cohort of treatment-naive PDAC patients enriched for exceptionally rare long-term survivors (LTS, n = 23; >5-year survival) alongside more common short-term survivors (STS, n = 24), enabling direct interrogation of mechanisms underlying durable tumour control. We develop a multi-scale spatial framework integrating multiplex imaging, computational modelling, and transcriptomics to quantify TLS architecture. By modelling TLSs as higher-order immune assemblies, we define their structural states, spatial organisation, assembly rules and tissue context-dependency within the tumour microenvironment. We show that long-term survival is associated with organised, spatially integrated TLSs exhibiting coordinated B and T cell zoning, whereas short-term survival is characterised by disorganised, Treg-enriched, and spatially isolated TLSs, despite similar immune cell abundance. These architectural differences align with transcriptional programmes: LTS tumours display coordinated lymphoid and NF-{kappa}B-driven chemokine signalling, while STS tumours exhibit inflammation uncoupled from immune organisation. Together, these findings demonstrate that durable anti-tumour immunity in PDAC is defined by coordinated transcriptional, cellular, and spatial organisation, rather than immune presence alone. This study provides a blueprint for structure-informed strategies to reprogram the tumour microenvironment and improve outcomes in PDAC. Statement of significanceIntegrating multiplex imaging, computational modelling and transcriptomics in treatment-naive PDAC enriched for exceptional survivors shows that durable tumour control is associated with organised TLS architecture rather than immune abundance alone, providing a framework for immune-architecture-based biomarkers and therapeutic reprogramming.

cancer biology↗

bioRxiv2026

Tissue-Specific Prevalence and Clonal Architecture of BRCA1/2 LOH-Inducing Chromosomal Aneuploidy

Germline pathogenic variants in BRCA1 and BRCA2 confer disproportionately elevated cancer risks in breast and ovarian tissues, yet the basis for this tissue specificity remains incompletely understood. Here, we integrate bulk-tumor aneuploidy analysis across 340,824 cancer cases from three independent cohorts (TCGA, ICGC PCAWG, and FoundationCore) with single-cell whole-genome sequencing from two independent studies to investigate whether tissue-specific patterns of chromosomal deletion contribute to this phenomenon. We find that breast and ovarian cancers are consistently enriched for deletions of chromosome arms 17q and 13q--harboring the BRCA1 and BRCA2 genes, respectively--relative to other solid tumor types, and that mutational timing analysis independently places these deletions among the earliest somatic events in these cancers. Phylogenetic reconstruction of single-cell data reveals that in pre-malignant breast tissue from germline BRCA1/2 carriers, chr17q and chr13q deletions appear as localized subclonal events within small clades against a largely diploid background. In established malignancies, these same deletions are found within dominant clonal lineages accompanied by widespread genomic instability--consistent with clonal sweeps originating from early deletion events. These findings suggest that breast and ovarian cellular environments confer a selective advantage for chr17q and chr13q deletions, providing a mechanism that may contribute to the tissue-specific cancer risk observed in gBRCA1/2 carriers.

cancer biology↗

bioRxiv2026

Aurora kinase A is a synthetic lethal target in FANCA-deficient cancers

Loss-of-function genomic alterations in FANCA occur across multiple cancer types, yet no molecularly tailored therapies have successfully exploited this potential vulnerability. Using complementary unbiased approaches, including a genome-wide CRISPR/Cas9 loss-of-function screen and a high-throughput drug screen in isogenic cancer cell-based models, we identified Aurora kinase A (AURKA) as a reproducible synthetic lethal target of FANCA-deficient cancers. Inhibition of AURKA induced chromosomal instability, micronucleation, and early G2/M arrest selectively in FANCA-deficient cells, consistent with an increased reliance on mitotic checkpoint control. Mechanistically, FANCA deficiency is associated with an elevated AURKA expression at both the transcriptomic and protein levels, and with an upregulation of mitotic spindle and G2/M checkpoint gene signatures. Analysis of large-scale cancer genomics datasets, including over 650,000 clinically sequenced tumors, confirms that FANCA is the most frequently altered Fanconi anemia pathway gene across cancers, and that Fanconi anemia-defective tumors exhibit an increased tumor mutational burden and genomic instability. Collectively, our findings point to AURKA inhibition as a promising precision treatment strategy in FANCA-deficient cancers and provide a rationale to further explore this strategy in the clinic.

cancer biology↗

bioRxiv2025

Hallmarks of adaptive immunity in a metastatic clear cell renal cell carcinoma (ccRCC) long-term elite survivor

Clear cell renal cell carcinoma (ccRCC) presents with metastatic disease in nearly one-third of patients, with a 5-year survival of only [~]10%. Although anti-angiogenic therapies and immune checkpoint inhibitors (ICIs) have improved outcomes, therapeutic resistance and immune-related adverse events limit durable responses. Exceptional long-term survivors provide a unique opportunity to uncover mechanisms of sustained anti-tumour immunity and guide therapeutic strategies. Here, we present a spatial and temporal dissection of adaptive immunity in an extraordinary 16-year ccRCC survivor, integrating single-cell sequencing, multiplex immunofluorescence, B and T cell receptor (BCR/TCR) repertoire analysis, and antibody profiling. We reveal persistent and spatially coordinated B and T cell clones over 16 years, indicating long-term immune memory and surveillance throughout tumour evolution. B cell analysis identified pronounced clonal expansions involving intrinsically autoreactive IGHV4-34 B cells undergoing ongoing germinal centre (GC) maturation in tumour-draining lymph nodes (dLNs) and spleen. These clones progressively lost autoreactivity while acquiring tumour-specificity through somatic hypermutation (SHM), consistent with clonal redemption. Redeemed B cells displayed enhanced antigen-presenting functions and localised to tertiary lymphoid structures (TLS), where they interacted with CD8 T cells, as inferred from doublet analysis. In parallel, persistent CD8+ effector T cell clones highlighted the contribution of long-lived lymphocytes to tumour control. Comparison of the primary tumour and pancreatic metastatic with paired tumour-free dLNs showed that TLS mirrored GC functionality, particularly for PD-1+ CD8+ T cells, while TLS maturation was stroma-dependent and impaired at the metastatic site. Finally, tumour-derived BCRs revealed antibody binding to tumour specific "public" antigens without cross-reactivity to normal tissue. Together, these findings establish clonal persistence, TLS engagement, and coordinated B-T cell immune surveillance as hallmarks of durable tumour control, providing a framework for next-generation antibody therapeutics and patient stratification in metastatic ccRCC. What is already known on this topic.Tertiary lymphoid structures within the tumour microenvironment (TME) and clonal expansions of CD8+ T cells are associated with better survival and improved response to immunotherapies in ccRCC. However, identifying markers of prolonged survival is challenging due to the rarity of long-term survivors (LTS), and the predominance of studies focused on T cell immunity, leaving B cell contribution almost unexplored. What this study adds.This study provides an integrated analysis of B and T cell responses in an exceptional 16-year LTS with metastatic ccRCC, revealing key immunologic features of sustained anti-tumour immunity. Beyond T cell dynamics, it highlights the multifaceted role of B cells as antigen presenting cells, antibody producers, and modulators of CD8+ T cell effector functions through direct interactions within TLS. Importantly, it reveals potential tumour-specific antigens driving humoral responses. How this study might affect research, practice or policy.By uncovering mechanisms of sustained tumour control, this work establishes a framework for identifying adaptive immune biomarkers of long-term survival and refining patient stratification in metastatic ccRCC. Multimodal assessment of B cell specificity and spatial interactions between re-educated B cells and CD8+ T cells within TLS introduces a new paradigm for understanding coordinated B and T cell responses. These insights have direct implications for antibody-based drug discovery and development of immunotherapies that harness the B-T cell axis.

cancer biology↗

bioRxiv2025

Comprehensive perturbation of transcription factors in human cardiomyocytes reveals the regulatory architecture of congenital heart disease

Transcription factors (TFs), including DNA binding proteins and epigenetic co-regulators, control the timing of gene programs during lineage commitment1. However, systematically defining the dynamic activities of the human genomes nearly 2000 TFs during development remains challenging2,3. Here, we use Perturb-Seq to map the transcriptional impact of nearly all human TFs and a subset of enhancers during cardiomyocyte differentiation. Our results suggest that developmental regulatory networks are distributed across highly connected hub TFs4-6 that are dynamic across lineage and time, rather than organizing into top-down hierarchies directed by "master regulators". We also identify distinct TF ensembles that regulate sequential decision points during cardiac cell-fate specification by coordinating lineage-specific activation with alternate lineage repression. Separately, analysis of TF-TF cooperativity uncovers a dynamic interaction between MEF2 family TFs7 and members of the Polycomb Repressive complex 18-10 to execute alternative fate repression. Building on these regulatory interactions, we construct a deep learning transformer model to accurately predict perturbed TFs driving altered regulatory networks in patient-derived transcriptomes. Together, our results define the regulatory network architecture of human lineage specification and provide a platform for predicting TF function and interpreting disease mechanisms.

genomics↗

bioRxiv2025

U2AF1 mutations rescue deleterious exon skipping induced by KRAS mutations

The mechanisms by which somatic mutations of splicing factors, such as U2AF1S34F in lung adenocarcinoma, contribute to cancer pathogenesis are not well understood. Here, we used prime editing to modify the endogenous U2AF1 gene in lung adenocarcinoma cells and assessed the resulting impact on alternative splicing. These analyses identified KRAS as a key target modulated by U2AF1S34F. One specific KRAS mutation, G12S, generates a cryptic U2AF1 binding site that leads to skipping of KRAS exon 2 and generation of a non-functional KRAS transcript. Expression of the U2AF1S34F mutant reverts this exon skipping and restores KRAS function. Analysis of cancer genomes reveals that U2AF1S34F mutations are enriched in KRASG12S-mutant lung adenocarcinomas. A comprehensive analysis of splicing factor/oncogene mutation co-occurrence in cancer genomes also revealed significant co-enrichment of KRASQ61R and U2AF1I24T mutations. Experimentally, KRASQ61R mutation leads to KRAS exon 3 skipping, which in turn can be rescued by the expression of U2AF1I24T. Our findings provide evidence that splicing factor mutations can rescue splicing defects caused by oncogenic mutations. More broadly, they demonstrate a dynamic process of cascading selection where mutational events are positively selected in cancer genomes as a consequence of earlier mutations.

cancer biology↗

bioRxiv2025

Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells

Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple sgRNA delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.

genomics↗

bioRxiv2024

An analysis of RNA quality metrics in human brain tissue

Human brain tissue studies have historically used a range of metrics to assess RNA quality. However, few large-scale cross-comparisons of pre-sequencing quality metrics with RNA-seq quality have been published. Here, we analyze how well metrics gathered before RNA sequencing (post-mortem interval (PMI) and RNA integrity number RIN) relate to analyses of RNA quality after sequencing (Percent of counts in Top Ten genes (PTT), 5 bias, and 3 bias) as well as with individual gene counts across the transcriptome. We conduct this analysis across four different human cortical brain tissue collections sequenced with varying library preparation protocols. PMI and RIN have a low inverse correlation, and both PMI and RIN show consistent and opposing correlations with PTT. Unlike PMI, RIN shows strong consistent correlations with measurements of 3 and 5 bias, and RIN also correlates with 3,933 genes across datasets, in comparison to 138 genes for PMI. Neuronal and immune response genes correlate positively and negatively with RIN respectively, suggesting that different gene sets have divergent relationships with RIN in brain tissue. In summary, these analyses suggest that conventional metrics of RNA quality have varying degrees of value, and that PMI has an overall minimal but reproducible effect on RNA quality.

neuroscience↗

bioRxiv2024

EML4-ALK variant-specific genetic interactions shape lung tumorigenesis

Oncogenic fusions of EML4 and ALK occur in [~]5% of lung adenocarcinomas. More than 15 EML4-ALK variants with distinct breakpoints within EML4 have been identified, but the functional differences between these variants remain poorly understood. Here we use CRISPR/Cas9 somatic genome editing to generate autochthonous mouse models of the two most common EML4-ALK variants, V1 and V3, and show that V3 is more oncogenic than V1. By integrating these models with multiplexed genome editing, we quantify the effects of 29 putative tumor suppressor genes on V1- and V3-driven lung cancer growth in vivo and show that many tumor suppressor genes have dramatically variant-specific effects on tumorigenesis. Analysis of a novel dataset representing the largest human EML4-ALK lung cancer cohort to date identified alterations in the genomic landscape depending on the EML4-ALK variant. These findings demonstrate functional heterogeneity among EML4-ALK variants, suggesting that EML4-ALK variants behave more like distinct oncogenes than a uniform entity. More broadly, these findings highlight the dramatic impact of oncogenic fusions partner proteins on tumor biology.

cancer biology↗

bioRxiv2024

Novel mark recapture surveys reveal stable pigmentation patterns but age-related melanism in the common frog Rana temporaria

Variations in melanin-based pigmentation patterns are frequent evolutionary adaptations in vertebrates and allow the study of phenotypic diversity across populations as well as individual recognition. Age-related changes in such pigmentation patterns have been demonstrated for several species but remain rarely explored in adult amphibians. For the wide-ranging European common frog (Rana temporaria) dorsal melanism was discussed in the context of UV protection at high altitude or as thermal adaptation at high latitude, yet patterns remain unquantified across most of its range or in terms of individual variation over time. We investigated dorsal pigmentation patterns in a wild population of R. temporaria in a lowland site in England which was the focus of a 7 year high-intensity mark recapture programme. We collected dorsal photographs using phone cameras and analysed pigmentation patterns using visual classifications and ImageJ image processing software. Pigmentation was highly variable between individuals but overall: (i) the number of melanin-based spots was largely stable or decreased over time as enlarging spots sometimes merged but never disappeared; (ii) the total melanin-based pigmented area increased significantly over time on average, albeit not for all individuals; and (iii) pigmentation was similar in males compared to females although the darkest individuals tended to be males. Our analysis reveals important patterns in pigmentation change and stability in this widespread species but the mechanisms and specific drivers remain unknown.

zoology↗

bioRxiv2024

MU-BRAIN: MUltiethnic Brain Rna-seq for Alzheimer INitiative

Alzheimers Disease (AD) exhibits a complex molecular and phenotypic profile. Investigating gene expression plays a crucial role in unraveling the diseases etiology and progression. Transcriptome data across ethnic groups lack, negatively impacting equity in intervention and research. We employed 565 brains across six U.S. brain banks (n=399 non-Hispanic Whites, n=113 Hispanics, n=12 African Americans) to generated bulk RNA sequencing from prefrontal cortex. We sought to identify cross-ancestry and ancestry-specific differentially expressed genes (DEG) across Braak stages, adjusting for sex, age at death, and RNA quality metrics. We further validated our findings using the Religious Orders Study/Memory Aging Project brains (ROS/MAP; n=1,095) and performed metanalysis (n=1,660). We conducted Gene Set and Variation and Enrichment analysis (GSVA; GSEA). We employed a machine-learning approach for phenotype prediction and gene prioritization to construct a polytranscriptomics risk score (PTRS) splitting our sample into training and testing sub-samples, either randomly or by ethnicity ("ancestry-agnostic" and "ancestry-aware", respectively). Lastly, we validated top DEG using single-nucleus RNA sequencing (snRNAseq) data. We identified several DEG associated with Braak staging: AD-known genes VGF (Padj =3.78E- 07) and ADAMTS2 (Pad j=1.21E-04) were consistently differentially expressed across statistical models, ethnicities, and replicated in ROS/MAP. Genes from the heat shock protein (HSP) family, e.g. HSPB7 (Padj =3.78E-07), were the top differentially expressed genes and replicated in ROS/MAP. Ethnic-stratified analyses prioritized TNFSF14 and SPOCD1 as top Hispanics DEG. GSEA highlighted "Alzheimer disease" (Padj =4.24E-06) and "TYROBP causal network in microglia" (Padj =1.68E-08) pathways. Up- and down-regulated genes were enriched in several pathways (e.g. "Immune response activation signal pathways", "Vesicle-mediated transport in synapse", "cognition"). Ancestry-agnostic and ancestry-aware PTRS effectively classified brains (AUC=0.77 and 0.73 respectively) and replicated in ROS/MAP. snRNAseq validated prioritized genes, including VGF (downregulated in neurons; Padj=1.1 E-07). This is the largest diverse AD transcriptome in post-mortem brain tissue, to our knowledge. We identified perturbated genes, pathways and network expressions in AD brains resulting in cross- ethnic and ethnic-specific findings, ultimately highlighting the diversity within AD pathogenesis. The latter underscores the need for an integrative and personalized approach in AD studies.

genetics↗

bioRxiv2024

Direct, bisulfite-free 5mC and 5hmC sequencing at single-cell resolution

We report the first direct sequencing methodologies for quantitative detection of 5mC and 5hmC at single-base resolution and single-cell level, termed scTAPS (for 5mC + 5hmC) and scCAPS+ (for 5hmC specifically). With [~]90% mapping efficiency, our methods accurately benchmark 5mC and 5hmC profiles in CD8+ T and mES cells, respectively. Notably, scCAPS+ revealed a global increase in 5hmC within the hippocampus of aging mice, both in neurons and in non-neurons.

genomics↗

bioRxiv2024

Shift in cellular crosstalk reveals the immunosuppressive environment in cholangiocarcinoma patients.

Background and aimsCholangiocarcinoma (CCA) is a deadly disease, and this cancer entity is characterized by an abundant stroma. The tumor microenvironment (TME) plays an important role in aggressive behavior and poor response to therapeutics; however, underlying pathways are unknown. MethodsTo fill this gap, we used multiplexed immunohistochemistry, high-dimensional phenotyping, and transcriptomics to analyze human CCA samples and identify cell cluster crosstalk in patients with a poor prognosis. ResultsOur findings confirmed the presence of Tregs and the lack of effector memory cells in the tumor. New findings are the spatiality of the effector memory cells being more present in the peripheral tissue, for some reason these immune cells fail to reach the tumor niche. We revealed cancer crosstalk with macrophages and stromal cells and identified responsible genes in the poor prognosis group. Amongst the responsible ligand pairs are GAS6-AXL belonging to the TAM family. We then identified VCAN-TLR2 to be present and influencing the ECM in a way to supports immune exhaustion. Last, EGFR-TGF-{beta} is expressed in macrophages and this finding is important in Tregs induction and blocking cytotoxic T cell function. ConclusionsThe multiple mechanisms leading to the exclusion of relevant immune cells needed for an anti-cancer response and mechanisms leading to active immune suppression are part of complex cell-cell crosstalk. This study provides a deeper insight into the immune exhausted phenotype in CCA.

cancer biology↗

bioRxiv2023

Tissue-resident NK cells support survival in pancreatic cancer through promotion of cDC1-CD8T activity

The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC) prevents tumor control and strategies to restore anti-cancer immunity (i.e. by increasing CD8 T cell activity) have had limited success. Here we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident NK (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypoactive NKG2D-ve NK population, phenotypically reminiscent of trNK cells, that supports CD8 T cell involvement. We show an equivalent population in human PDAC cohorts that represents immunomodulatory trNK cells that could similarly support CD8 T cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5i/PD1 and IR-induced damage as a novel therapeutic approach.

cancer biology↗