Cancer Cell

Bulk and single-cell analyses of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) have revealed a largely immunosuppressive milieu. Thus far, efforts to utilize insights from TME features to facilitate more effective therapeutics have largely failed. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy samples (n=22) and surgical samples (n=6), integrated with 3 public datasets (n=49), resulting in [~]140,000 individual cells from 77 patients. Based on expression markers assessed by Seurat v3 and differentiation status assessed by CytoTRACE, we divided the resulting tumor cellular clusters into 5 molecular subtypes based on expression of previously reported marker genes: Basal, Mixed Basal/Classical, Classical Low, Classical High, and ADEX. We then queried these 5 tumor cell profiles, along with 15 scRNA-seq-derived tumor microenvironmental cellular profiles, in 391 bulk expression samples from 4 published datasets of localized PDAC with associated clinical metadata using CIBERSORTx. Through unsupervised clustering analysis of these 20 cell state fractions representing tumor, leukocyte and stromal cells, we identified 7 unique clustering patterns representing combinations of tumor cellular and microenvironmental cell states present in PDAC tumors. We termed these cell state patterns communities, and found them to correlate with overall survival, tumor ecotypes, and tumor cellular differentiation status. The community associated with worst overall survival contained basal tumor cells, exhausted CD4 and CD8 T cells, and was enriched for fibroblasts. In contrast, the highest overall survival was associated with a community high in immune cell enrichment. The differentiation state of tumor cells (assessed by CytoTRACE) was also correlated with survival in a dose-dependent fashion. Further, we identified a subset of PDAC samples that were significantly enriched for CD8 T and plasma cells that achieved a 2-year overall survival rate of 71%, suggesting we can identify PDAC patients with significantly improved prognoses and, potentially, higher sensitivity to immunotherapy. In summary, we identified novel tumor microenvironmental communities from high-dimensional analysis of PDAC RNA sequencing data that reveal new connections between tumor microenvironmental composition and patient survival that could lead to better upfront risk stratification and more personalized clinical decision-making.

Non-small cell lung cancer (NSCLC) patients exhibit heterogeneous responses to immunotherapy (IT) with high resistance rates, highlighting the need for precise biomarkers predictive of treatment outcomes. In a prospective cohort study, we longitudinally assessed liquid biopsy samples from NSCLC patients undergoing IT at four distinct time points (T1 pre-treatment, T2 post-second cycle, T3 six months, and T4 one year). We profiled plasma-derived cell-free DNA methylation and extracellular vesicle-associated microRNAs from 79 metastatic NSCLC patients treated with immune checkpoint inhibitors (ICIs). High-dimensional omics data were integrated using Multi-Omics Factor Analysis (MOFA2) to uncover latent molecular subtypes, which we termed MOFA-Derived Clusters (MDCs), independently established at baseline (MDC-T1) and post-second cycle (MDC-T2). Differential expression and methylation analyses, pathway enrichment, and immune phenotyping via flow cytometry were used to characterize the molecular and immunological landscape of each MDC. External validation was performed using independent NSCLC cohorts for miRNAs (Genova et al., 2024, n=54) and methylation (SMC-Cohort, GSE119144, n=57). MDCs captured divergent survival outcomes and reflected biologically coherent processes including angiogenesis, cytoskeletal remodeling, and immune signaling. Projection of MDCs onto later time points (T3, T4) supported the temporal relevance of early molecular signatures. MDCs also displayed immunological correlates via circulating immune cell subsets. Importantly, MDC classifiers demonstrated consistent survival stratification in external cohorts, particularly MDC-T2. This study defines a multi-omic, liquid biopsy-based framework for molecular subtyping in NSCLC to manage ICI treatment. Our MDC signatures reveal clinically meaningful, treatment-informative biology and offer a path toward minimally invasive patient stratification in immuno-oncology.

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central role in cellular signaling. Despite this, their promise remains largely untapped due to the technical challenges inherent to profiling cell surface proteins. Here, we employed N-glycoproteomics to analyze 85 patient-derived xenografts (PDX), constructing Glyco PDXplorer - an in vivo pan-cancer atlas of cancer-derived cell surface proteins. We developed a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validated FAT2 as a head and neck squamous cancer (HNSC) enriched surface protein with limited expression in normal tissue. Functional studies revealed that FAT2 is essential for HNSC growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR) T cells targeting FAT2 demonstrated potent anti-tumor activity in HNSC models. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal resource to inform therapeutic target discovery for multiple cancers. HIGHLIGHTSO_LIPan-cancer landscape of cancer-derived cell surface proteins detected in vivo C_LIO_LIDevelopment of a multi-omic discovery pipeline to prioritize proteins with optimal expression profiles as immunotherapy targets C_LIO_LIIdentification and validation of FAT2 as a head and neck squamous cancer enriched surface protein with minimal expression in normal tissues C_LIO_LIFAT2 coordinates cell surface organization, adhesion, growth and survival through the integrin-PI3K-AKT pathway C_LIO_LIFAT2 CAR T cells demonstrate anti-tumour activity in pre-clinical models C_LI

Regional lymph nodes (LNs) in the thoracic cavity serve as essential immunological hubs that coordinate humoral and cell-mediated responses against the development and progression of non-small cell lung cancer (NSCLC). To investigate immune dysregulation in the non-metastatic regional LNs of patients with aggressive NSCLC, we performed multimodal profiling on 36 LNs from 11 patients undergoing curative-intent resection including CITE-seq, scRNA-seq, and Imaging Mass Cytometry (IMC). Regional N1 LNs from patients with more aggressive disease (stage IB-IIIA) exhibited a significant enrichment of dysfunctional CD8 T cells and regulatory T cells (Tregs) compared to N2 LNs and LNs from patients with less aggressive disease (stage IA). These immune subsets were spatially co-localized with mature regulatory dendritic cells (mregDCs; CD1c, TIM3, LAMP3), forming an immunosuppressive niche uniquely enriched in the N1 LNs of higher-stage patients. Concurrently, higher-stage N1 LNs contained larger number of "decorticated" B-cell follicles characterized by decreased encapsulation of the mantle zone layer surrounding the germinal centers. This mantle zone disorganization was associated with increased spatial niches involving Tregs, CD68+ CD163 TIM3 Macrophages, CD163 TIM3dim Monocytic-Myeloid Derived Suppressor Cells (M-MDSC), plasma B cells, and a decrease in spatial niches involving CD4 T helper cells and fibroblastic reticular cells (FRCs). Together, our findings reveal parallel alterations in humoral and cell-mediated immunity within the regional LNs of patients with aggressive NSCLC.

Many cancer patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Circulating biomarkers have the potential to identify patients with primary resistance or early progression on therapy to alter treatment course and avoid unnecessary toxicity. Unbiased multimodal proteomic profiling in blood has been underexplored due to the previously limited scalability of multiplexing technologies or cohorts lacking time-series sampling. To address this, we performed plasma proteomic profiling of >2,900 proteins and high-dimensional mass cytometry of peripheral blood lymphocytes across serial time points in 250 metastatic melanoma patients on ICB treatment. We further obtained 92 patient-matched tumor samples, which were processed for single-cell and/or bulk RNA sequencing. Proteins upregulated post-ICB were associated with inflammatory pathways involving the activation of effector immune functions. Expression of genes corresponding to these proteins was higher in immune cells involved in recruitment and tumor reactivity. Expression of genes corresponding to plasma proteins more abundant in non-responders was highest in suppressive myeloid subsets and malignant cells. We further posit the involvement of these non-responder genes in immunosuppressive and pro-tumor interactions, which we confirmed using publicly available spatial transcriptomic data. We also found that epithelial-specific proteins in the circulation of responders post-ICB correlate with patient toxicity and likely originate from healthy tissues. Together, these data represent one of the deepest peripheral biomarker studies using paired blood and tumor samples in melanoma patients treated with ICB, and begin to elucidate the complex interplay between tumors and the systemic immune response within the host.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related death despite the availability of therapies targeting the tumor microenvironment (TME) and/or the tumor. Immunotherapy and novel targeted therapy have improved the management of NSCLC, but the heterogeneity of the disease still hampers treatment efficacy as well as further therapeutic advances. Here, we used spatial single-cell data complemented with bulk RNAseq and whole exome sequencing to investigate the TME of 192 resected, mostly early-stage NSCLC tumors and to characterize associations of genetic, clinical and lifestyle factors with cellular and histological properties of the TME. We found, that the TME of squamous cell carcinoma (LUSC) harbored stronger signs of inflammation and immune exhaustion than adenocarcinoma (LUAD), but interestingly that elevated PD-L1 expression was associated with inflammation and markers of lymphocyte activation/exhaustion only in LUAD. Smoking correlated with T cell infiltration and TP53 mutation in LUAD, and TP53 mutation was associated with proliferation of tumor cells. In both histologies, naive and BCL2+ T cells decreased with higher clinical stages. EGFR-driven tumors showed fewer proliferating, activated and exhausted T cells, but more CD4 T cells and HLA-DR+ tumor cells, than tumors with wild type EGFR. Multi-modal integration of our four data types showed that most variation in our cohort was along the axes of histology, smoking, TP53 mutation, and inflammation. Our integration identified histology-specific prognostic signatures, with a LUSC-enriched profile of proliferation, inflammation, and smoking associated with poor prognosis in LUAD. In summary, we provide a rich, multimodal, single-cell characterization of a large NSCLC cohort as a resource and suggest that future investigations of biomarkers for ICI in NSCLC will benefit from stratification by histology.

Ide-cel and cilta-cel are the two FDA-approved anti-BCMA CAR T cell therapies for the treatment of relapsed/refractory multiple myeloma. Here, we studied a patient who was initially treated with ide-cel with progressive disease and subsequently treated with cilta-cel with a complete response. To elucidate the underlying mechanisms underpinning the distinct clinical outcomes, we conducted multimodal, cross-tissue, and longitudinal single-cell analyses. This enabled us to directly compare the specific cellular and molecular factors distinguishing these two CAR therapies including their cell phenotypes, post-infusion kinetics, and endogenous immune landscapes. We found that the ide-cel infusion product was dominated by CD4+ CAR T cells, upregulated a terminal effector phenotype, and exhibited elevated activation signatures. Post-infusion, ide-cel CAR T cells failed to proliferate, sustain cytotoxicity, or migrate into the bone marrow, resulting in persistent myeloma cells and dysregulated monocytes and natural killer cells. In contrast, the cilta-cel infusion product exhibited a balanced ratio of CD4+ and CD8+ CAR T cells, upregulated a resident memory-like signature, and displayed signatures of IL-1 and IL-2 family cytokine signaling. Post-infusion, cilta-cel CAR T cells retained their resident memory-like profile, were durably retained in the peripheral blood, and successfully infiltrated the bone marrow, leading to effective tumor clearance and reestablishment of immune homeostasis. Our results present important clinical evidence that cilta-cel can serve as an effective salvage treatment following ide-cel failure. By providing a direct patient-matched comparison between two CAR therapies, our study uncovers important insights into both CAR T-cell intrinsic properties and immune environmental factors that contribute to effective BCMA CAR T-cell treatment.

Immune checkpoint inhibitor immunotherapy has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite the immense success, still a significant proportion of patients do not develop durable responses, allowing disease progression accompanied by high mortality rates. Therefore, there is an imperative need for identification of reliable non-invasive predictive biomarkers to guide therapeutic decisions. Herein, we constructed a blood immunomap in NSCLC patients with metastatic disease, using a high-dimensional mass cytometry approach. Assessment of clinical responses to aPD1 immunotherapy revealed, among others, a significant expansion of CD8+PD-L1+ T cells in individuals not responding to immunotherapy. Of interest, CD8+PD-L1+ T cells were enriched in tumor biopsies and bronchoalveolar lavage of NSCLC individuals at early stages of disease as well as in pleural infusions of individuals with thoracic malignancies. Transcriptomic analysis revealed that CD8+PD-L1+ T cells exhibited a regulatory/exhausted phenotype, while various transcripts associated with the overall survival of NSCLC individuals, were mapped. Overall, our findings define an immunomap in the early stage and advanced NSCLC patients and identify immune-related events which may benefit the quest for identification of predictive biomarkers of immunotherapy responses.

Preclinical cancer models often use subcutaneous (SC) implantation, which fails to recapitulate the native tumor microenvironment (TME) of orthotopic (ORT) sites. To resolve these differences, we used single-cell RNA sequencing (scRNA-seq) on paired SC and ORT implants of the CKP syngeneic gastric cancer model. Histopathological differences were minimal, but scRNA-seq revealed profound TME divergence. ORT tumors displayed robust stromal activation, coordinated fibroblast and endothelial signaling, and an immune compartment marked by higher T/NK cell activation and IgA- biased B cell plasma programs, reflecting a physiological mucosal environment. In contrast, SC tumors had higher overall T cell infiltration but showed markedly increased CD8+ T cell exhaustion and an enriched oxidative tumor program. Our findings provide critical guidance: SC models are optimal for high-throughput and exhaustion-focused assays, whereas ORT models are indispensable for studying organ-specific immune and stromal biology with translational fidelity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/680400v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@bfa6c2org.highwire.dtl.DTLVardef@5d28aforg.highwire.dtl.DTLVardef@1a38f2org.highwire.dtl.DTLVardef@190bcd9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The addition of aPD1 to 5-FU/platinum in advanced gastric cancer (GC) yields variable responses. To understand cooperativity between chemotherapy and immunotherapy, we previously reported a phase II trial sequentially adding pembrolizumab to 5-FU/platinum. In this study, we use single-cell RNA- and TCR-sequencing to analyze 66,813 T cells from primary tumor biopsies pre-treatment, post-chemotherapy, and post-immunotherapy in 33 patients. We observed greater abundance, persistence, and recruitment of T cells with predicted tumor-reactivity in patients with prolonged progression-free survival (slow progressors). Increased B cell abundance and predicted B cell to T cell interactions supported T cell memory and co-stimulation, providing a mechanism for increased abundance and persistence of progenitor-exhausted and tumor-reactive T cells in slow progressors. T cell clones emerging in the tumor after immunotherapy were in the blood before treatment only in slow progressors. Our study thus highlights pre-treatment and early chemotherapy-induced T cell dynamics and B cell to T cell interactions that may drive durable response to chemoimmunotherapy in GC.

In syngeneic murine breast cancer models, poly(ADP-ribose) polymerase inhibitor (PARPi) and anti-PD-L1 combinations induce deep, sustained responses independent of BRCA1 or BRCA2 mutation (BRCAm) status. We therefore investigated this combination in the AMTEC clinical trial, in which a one-month olaparib run-in was followed by combined olaparib and durvalumab in participants with non-BRCAm metastatic triple negative breast cancer. To characterize adaptive responses to olaparib monotherapy, paired biopsies taken before and during the PARPi lead-in were deeply characterized by DNA, RNA, and protein multi-omic analyses, including spatially resolved single-cell proteomics for tumor and immune contexture. We identified multiple potential tumor-intrinsic and microenvironmental biomarkers from pre-treatment and on-olaparib biopsies that robustly predicted participant response to combined olaparib and durvalumab. Notably, the on-olaparib biopsy provided the greatest information content, suggesting that early adaptations of malignant and immune cells to PARPi can serve as a predictor of potential benefit from combined PARPi and anti-PD-L1 therapy.

Targeted immunotherapies against B-cell maturation antigen (BCMA) have transformed the treatment landscape of Multiple Myeloma (MM). Fc receptor-like 5 (FCRL5) has emerged as an alternative target. However, resistance frequently emerges within months, posing a significant clinical challenge. Structural alterations and mutations in BCMA only account for the minority of cases and insights into BCMA antigen escape remain largely unknown. This study investigates novel (epi)genetic mechanisms of antigen escape through comprehensive multi-omic Oxford Nanopore profiling of sequential pre-treatment and relapse samples. We identify acquired DNA-hypermethylation across the entire BCMA gene, and hypermethylation of the FCRL5 promoter, both resulting in epigenetic gene silencing as novel resistance mechanisms through which MM cells evade therapy. These findings underscore the dynamic clonal evolution of MM under therapeutic pressure and highlight the critical role of epigenetic modifications in resistance. Furthermore, we demonstrate the potential of advanced sequencing technologies for capturing epigenetic and complex genomic alterations in clinical settings, paving the way for personalized treatment strategies and predictive biomarkers for early resistance detection. Statement of significanceAcquired DNA hypermethylation of BCMA and FCRL5 regulatory regions, leading to gene expression downregulation, represent novel epigenetic resistance mechanisms to anti-BCMA and anti-FCRL5 immunotherapies. Furthermore, DNA methylation marks serve as a molecular memory of therapeutic pressure, capturing the treatment history of cancer cells.

Anaplastic thyroid cancer (ATC) is one of the most lethal human cancers, with some patients succumbing to the disease within weeks of diagnosis. Although a subset of patients with ATC with BRAFV600E mutation respond to the monomeric type I RAF inhibitor (RAFi) dabrafenib in combination with MEK inhibitor (MEKi) trametinib, most rapidly develop adaptive or acquired resistance. These patients, along with those who do not harbor the BRAFV600E alteration, have limited treatment options. To understand the mechanism of resistance to dabrafenib and trametinib, we utilized multi-region whole genome, high-coverage whole exome and single nuclei RNA-sequencing of ATC patient tumours to unravel genomic, transcriptomic, and microenvironmental evolution during type I RAFi and MEKi therapy. Single-cell nuclei RNA sequencing of matched primary and resistant ATC patient tumours identified reactivation of the MAPK-pathway, along with immunosuppressive macrophage proliferation, underlying the development of acquired resistance. Our translational genomics led us that hypothesize that type II RAFi, which inhibit both RAF monomers and dimers, can be efficacious in overcoming treatment resistance. Screening of a panel of type II RAFi revealed that ATC cell lines are exquisitely sensitive to the type II RAFi, naporafenib, by inhibiting EphA2-mediated MAPK-signaling. We further demonstrated that naporafenib, in combination with the MEKi trametinib, can durably and robustly overcome both innate and acquired treatment resistance to dabrafenib and trametinib using ATC cell lines and patient-derived xenograft models. Finally, we describe a novel mechanism of acquired resistance to type II RAFi and MEKi through compensatory mutations in MAST1. Taken together, our work using translational and functional genomics has unraveled the differential mechanisms of treatment resistance to type I and type II RAFi in combination with trametinib and rationalizes the clinical investigation of type II RAFi in the setting of thyroid cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with a 5-year survival rate of less than 10%. Chemotherapy is the current standard-of-care (SOC) for advanced PDAC; however, treatment resistance driven by a complex and immunosuppressive tumor microenvironment (TME) limits its effectiveness. To address mechanisms of resistance, we studied cabozantinib (cabo), a multi-kinase inhibitor approved for several solid tumors. Cabo has shown promise in preclinical PDAC models and clinical trials, particularly when combined with immunotherapy, but its mechanisms of action within the human PDAC TME and its potential to overcome therapy resistance remain unclear. Using CosMx Spatial Molecular Imaging (CosMx SMI) and Orion Multiplex Immunofluorescence (MxIF), we analyzed PDAC tissues collected after first line systemic chemotherapy and the Whipple surgical procedure, as well as from liver or lung metastatic sites from patients with PDAC. To functionally model the TME, we established matched patient-derived organoid (PDO) co-cultures harboring cancer associated fibroblasts (CAFs), and autologous immune cells (IMM) (PDO/CAF/IMM). These models were used to evaluate the effects of cabo and pembrolizumab, an anti-PD1 immune checkpoint inhibitor, with benchmarking of findings to the patients TME. Spatial analysis of post-chemotherapy and metastatic PDAC tissues revealed heterogeneous cellular neighborhoods within the TME, including enrichment of Schwann cells, CAFs, T regulatory cells, and cancer stem cells (CSCs). Distinct niches were observed in metastatic liver tissues, characterized by mesenchymal stem cells, fibroblasts, including myCAFs and iCAFs, and CSCs expressing CD44 and TROP2. In PDO/CAF/IMM co-cultures, treatment with cabo in combination with pembrolizumab enhanced cancer cell death by depleting myeloid-derived suppressor cells (MDSCs) and promoted cytotoxic T lymphocyte proliferation. Across both patient tissues and treated co-cultures, a persistent SOC-resistant cancer stem cell population emerged that expressed CD44 variant 9 (CD44v9). Taken together, these integrative spatial and organoid-based studies demonstrate that cabo can remodel the PDAC TME and potentiate PD-1 immunotherapy in preclinical models, while resistance associates with a CD44v9+ CSC population, revealing a potential therapeutic target.

The advent of combination immune checkpoint inhibitors (cICI), specifically ipilimumab and nivolumab, has transformed the treatment landscape for metastatic renal cell carcinoma (mRCC), offering durable remissions and improved outcomes for intermediate- and poor-risk patients. However, intrinsic resistance remains a significant challenge, with 40-60% of patients failing to achieve meaningful responses. Here, we conducted a comprehensive, multi-omic analysis of systemic and tumor-associated immune responses in mRCC patients enrolled in a clinical trial (CA209-980, SAKK07/17, NCT03297593) testing a novel response-adapted cICI regimen. Our study aimed to identify immune correlates of response to cICI therapy. High-dimensional mass cytometry and single-cell proteomic and bulk RNA sequencing of the tumor revealed an enrichment of Th17 CD4+ T cells in responders. These cells exhibited upregulated IL-21-driven pathways, IL-17 signaling, and inflammasome-associated processes, highlighting their central role in therapeutic efficacy. Our integrative analysis underscores the importance of Th17 cells in mediating effective responses to cICI and provides a framework for developing predictive biomarkers and therapeutic strategies to overcome resistance. These findings highlight the translational potential of targeting Th17-centric pathways to enhance immunotherapy outcomes in mRCC and potentially other cancers. This work represents a significant step toward advancing personalized oncology through integrated immune profiling.

Recent advancements in cancer immunotherapy have improved patient outcomes, yet responses to immunotherapy remain moderate. We conducted a Phase II clinical trial (NCT04718415) involving 51 cancer patients undergoing neoadjuvant chemoimmunotherapy and applied single-cell RNA and T/BCR sequencing on tumor and blood samples to elucidate the immune cell perturbations. Our findings associate poor response with reduced levels of CCR7+CD4 Naive T cells and CD27+ Memory B cells, as well as higher expression of immunosenescence-related genes in T and B cell subsets. Using naturally aged and Ercc1+/- transgenic aging mouse models, we found that senolytics enhance the therapeutic efficacy of immunotherapy in multiple solid tumors by mitigating tumor immunosenescence. Notably, we launched a Phase II clinical trial, COIS-01 (NCT05724329), which pioneers the combination of senolytics with anti-PD-1 therapy. The clinical results demonstrate that this therapeutic strategy is associated with a favorable safety profile and therapeutic efficacy, significantly mitigating adverse effects and alleviating immunosenescence. These findings underscore the pivotal role of immunosenescence characteristics in influencing the effectiveness of immunotherapy and suggest a promising therapeutic efficacy along with a beneficial safety assessment for the combination of senolytics with anti-PD-1 therapy.

Glioblastoma is a highly aggressive cancer associated with a dismal prognosis, with a mere 5% of patients surviving beyond five years post-diagnosis. Current therapeutic modalities encompass surgical intervention, radiotherapy, chemotherapy, and immune checkpoint inhibitors (ICB). However, the efficacy of ICB remains limited in glioblastoma patients, necessitating a proactive approach to anticipate treatment response and resistance. In this comprehensive study, we conducted a rigorous analysis involving two distinct glioblastoma patient cohorts subjected to PD-1 blockade treatments. Our investigation unveiled that a significant portion, 60%, of patients exhibit persistent disease progression despite ICB intervention. To elucidate the underpinnings of resistance, we characterized the immune profiles of glioblastoma patients with continued cancer progression following anti-PD1 therapy. These profiles revealed multifaceted defects, encompassing compromised macrophage, monocyte, and T follicular helper responses, impaired antigen presentation, aberrant regulatory T cell (Tregs) responses, and heightened expression of immunosuppressive molecules (TGFB, IL2RA, and CD276). Building upon these resistance profiles, we leveraged cutting-edge machine learning algorithms to develop predictive models and accompanying software. This innovative computational tool achieved remarkable success, accurately forecasting the progression status of 82.82% of glioblastoma patients following ICB, based on their unique immune characteristics. In conclusion, our pioneering approach advocates for the personalization of immunotherapy in glioblastoma patients. By harnessing patient-specific attributes and computational predictions, we offer a promising avenue for the enhancement of clinical outcomes in the realm of immunotherapy. This paradigm shift towards tailored therapies underscores the potential to revolutionize the management of glioblastoma, opening new horizons for improved patient care.

Immunotherapy resistance remains a major challenge in immuno-oncology. We have previously demonstrated that a tumor-intrinsic NLRP3 inflammasome signaling pathway promotes adaptive immunotherapy resistance by inducing the recruitment of granulocytic myeloid-derived suppressor cells. We now confirm that elevated tumor NLRP3 signaling activity correlates with checkpoint inhibitor resistance in several independent cohorts of stage III and IV melanoma patients, as well as in advanced gastroesophageal (GE) adenocarcinoma patients. In situ hybridization studies further demonstrate that tumor NLRP3 copy-number gain is observed in immunotherapy resistant melanomas and GE adenocarcinomas harboring enhanced NLRP3 signaling activity. Spatial transcriptomic analysis of GE adenocarcinoma tissues reveals that NLRP3 signaling activity inversely correlates with NLRC5 and MHC class I-associated gene expression. Indeed, pre-clinical models of melanoma and GE adenocarcinoma demonstrate that Nlrp3 amplification suppresses NLRC5-mediated MHC class I upregulation, while pharmacologic NLRP3 inhibition augments tumor MHC class I surface levels. Upon activation, tandem mass spectrometry-supported signaling studies reveal that NLRP3 binds to and inhibits STAT1 dimerization, nuclear translocation, and NLRC5 transcription. Consistent with these findings, inhibiting the NLRP3 inflammasome augments tumor STAT1-NLRC5 signaling and overcomes anti-PD-1 resistance in an orthotopic model of chromosomal instability (CIN) gastric adenocarcinoma. This work indicates that the tumor NLRP3 inflammasome signaling pathway merits further clinical study as a therapeutic target and a source of companion biomarkers for overcoming checkpoint inhibitor resistance in cancer patients.

Unprecedented advantages in cancer treatment with immune checkpoint inhibitors (ICI) remain limited to a subset of patients. Systemic analyses of the regulatory 3D genome architecture linked to individual epigenetics and immunogenetic controls associated with tumour immune evasion mechanisms and immune checkpoint pathways reveals a highly prevalent patient molecular profiles predictive of response to PD-(L)1 immune checkpoint inhibitors. A clinical blood test based on the set of 8 3D genomic biomarkers has been developed and validated on several independent cancer patient cohorts to predict response to PD-(L)1 immune checkpoint inhibition. The predictive 8 biomarker set is derived from prospective observational clinical trials, representing 229 treatments with Pembrolizumab, Atezolizumab, Durvalumab, in diverse indications: melanoma, non-small cell lung, urethral, hepatocellular, bladder, prostate cancer, head and neck, vulvar, colon, breast, bone, brain, lymphoma, larynx cancer, and cervix cancers. The 3D genomic 8 biomarker panel for response to immune checkpoint therapy achieved high accuracy up to 85%, sensitivity of 93% and specificity of 82%. This study demonstrates that a 3D genomic approach could be used to develop a predictive clinical assay for response to PD-(L)1 checkpoint inhibition in cancer patients.

BackgroundPost-translational modifications (PTMs) represent a fourth dimension of the genetic code, orchestrated by the Golgi apparatus and central to the biology of cancer. The prevailing paradigm of oncofetal reprogramming posits that cancer cells reactivate embryonic developmental programs to drive tumorigenesis; however, the lineage-specific nature of this reversion remains incompletely defined. This review advances and systematically evaluates the hypothesis that the cancer PTM landscape is a traceable relic of the cells embryonic germ layer origin, ectoderm, mesoderm, or endoderm--offering a novel, developmentally-informed framework for precision oncology. ObjectivesTo systematically review and synthesize the global evidence linking cancer PTMs to their developmental origins and to evaluate the efficacy, safety, and implementation of PTM-targeted therapeutics through this novel developmental lens, with LLM assistance. MethodsFollowing PRISMA 2020 guidelines, and using LLM assistance, a systematic search of PubMed, Embase, Web of Science, Cochrane Library, and extensive grey literature sources, including over 3,000 theses, dissertations, clinical trial registries, and institutional reports, from inception to June 2025 was conducted. ResultsFrom an initial screen of over 25,000 records, 3,128 studies met the inclusion criteria, encompassing data from over 500,000 patients. Our analysis revealed distinct, germ-layer-specific PTM signatures and corresponding therapeutic vulnerabilities. Ectoderm-derived cancers (e.g., neuroblastoma, melanoma) are characterized by aberrant oncofetal glycosylation. Anti-GD2 immunotherapy, which targets a neural crest-specific glycan, demonstrated a profound survival benefit in high-risk neuroblastoma (Hazard Ratio for overall survival: 0.57, 95% CI 0.42-0.78, p<0.001). Mesoderm-derived malignancies (e.g., sarcomas, leukemias) exhibit dysregulated phosphorylation and SUMOylation. These cancers respond preferentially to kinase inhibitors (HR for overall survival: 0.72, 95% CI 0.68-0.76, p<0.001) and cellular therapies like CAR-T, which achieve overall response rates exceeding 80% in hematologic malignancies. Endoderm-derived adenocarcinomas (e.g., lung, colorectal) display a heightened dependency on the ubiquitin-proteasome system for managing proteotoxic stress, validating proteasome inhibitors (HR for overall survival: 0.77, 95% CI 0.71-0.84, p<0.001) and emerging PROTACs as key therapeutic classes. Implementation science analysis revealed profound global disparities, with treatment costs exceeding $450,000 per sequence and access to advanced PTM therapies below 5% in low-income countries. ConclusionThe post-translational modification landscape of cancer is fundamentally imprinted by its embryonic lineage, with the Golgi apparatus acting as a key arbiter of this oncofetal memory. This developmental framework provides a powerful new tool for rational drug design, biomarker discovery, and patient stratification. However, translating this scientific progress into global patient benefit requires timely and coordinated policy action to address the profound implementation chasm created by prohibitive costs and systemic inequities in healthcare access.