Ascites in peritoneal metastases (PM) portends a poor prognosis. However, molecular ligands within ascites and their biological relevance remain unclear. A comprehensive multiomic analysis, including proteomic analysis of 397 malignant cell-free ascites representing 21 histological origins of PM, was performed to decipher pertinent biological themes, identify and validate therapeutically relevant paracrine factors in cell-free ascites, and unravel cell-specific secretion of key paracrine factors. We further developed a nanobody targeting PAI-1 and tested it in vitro, in vivo and ex vivo human explant models. Mass spectrometry analysis identified multiple biological themes including the coagulation cascade. New and previously identified paracrine factors amenable to therapeutic perturbation were also unraveled. Single-cell transcriptomic analysis confirmed secretion of two functionally antagonistic regulators of fibrinolysis (S100A10 and PAI-1) by distinct cell populations in PM ascites, suggesting upregulation of these ligands are due to active secretion by cells rather than homeostatic regulation. We also demonstrated proof of concept of efficacy with minimal toxicity of our nanobody against PAI-1. Collectively, this study explores the biological significance of proteins in malignant cell-free ascites in PM and introduces the therapeutic potential of targeting PAI-1 via a nanobody.
Lineage plasticity drives treatment resistance by enabling tumors to shed their cellular identity. Androgen receptor (AR)-negative castration-resistant prostate cancer (CRPC) is characterized by loss of AR signaling associated with extensive epigenetic barriers and repressive chromatin topology, yet its regulatory mechanisms remain poorly characterized. We combined public transcriptomic and epigenomic analyses with RNA-seq, ATAC-seq, RNAi-mediated depletion, low-dose pharmacological inhibition, ChIP-qPCR, chromosome conformation capture, methylation, reporter and therapeutic-response assays in AR-low/null prostate cancer cell lines and xenograft models, together with CRPC patient-derived organoids. RNAi-mediated depletion or low-dose pharmacological inhibition of KIF11 was associated with activation of AR-associated transcriptional programs in AR-low/null prostate cancer models. KIF11 inhibition was accompanied by increased chromatin accessibility and higher FOXA1 and GRHL2 occupancy at the AR enhancer, together with increased EP300 and SUPT5H occupancy at AR regulatory elements. Concurrently, KIF11 inhibition reduced DNA methylation at CTCF motifs, increased CTCF occupancy, and enhanced enhancer–promoter communication at the AR locus. This topological resetting established a stable AR transcriptional program and feed-forward reinforcement circuit. Consequently, this switch drove a lineage return from AR-low/null androgen-insensitive prostate cancer models to an AR-responsive transcriptional state, conferring sensitivity to enzalutamide and apalutamide across cell-line, organoid, and xenograft models. These findings identify KIF11 inhibition as a lineage-remodeling strategy that induces AR-responsiveness in AR-low/null androgen-insensitive prostate cancer models through enhancer–promoter loop construction. Low-dose, non-cytotoxic KIF11 inhibition activates androgen receptor signaling and sensitizes otherwise antiandrogen-insensitive models to enzalutamide and apalutamide, providing an approach to overcome lineage plasticity-driven therapeutic resistance.
Cancer-associated fibroblasts (CAFs) play a key role in stromal remodeling, immune exclusion, and therapeutic resistance in lung adenocarcinoma (LUAD). However, CAF heterogeneity has largely been defined by static phenotypic classifications, leaving unresolved whether tumor cells actively instruct fibroblast differentiation toward pathogenic states that drive immune evasion and immunotherapy resistance. Specifically, the mechanisms by which tumor-stroma communication programs CAF functional states remain poorly understood. We performed integrative single-cell RNA sequencing analyses to create a detailed stromal atlas of LUAD and elucidate fibroblast lineage trajectories. Mechanistic drivers of CAF programming were examined using tumor–fibroblast co-culture systems, extracellular vesicle (EV) isolation and functional fractionation, and quantitative EV proteomics. Causal signaling pathways were confirmed through genetic and pharmacologic perturbations. Clinical relevance was evaluated across multiple independent patient cohorts and by multiplex immunofluorescence in LUAD specimens. Therapeutic implications were tested in immunocompetent mouse tumor and metastasis models, including combination treatments with PD-1 blockade. Single-cell analysis showed that fibroblasts in LUAD follow a continuous differentiation path toward a myofibroblast-like state marked by MMP11 expression. This MMP11⁺ CAF subset exhibited a transcriptional program associated with extracellular matrix remodeling, collagen organization, and immune exclusion, and was correlated with poor clinical outcomes. Tumor cells had varying abilities to trigger this CAF program, which was primarily mediated by tumor-derived EVs rather than soluble factors. Comparative EV proteomics identified secreted phosphoprotein 1 (SPP1) as a selectively enriched cargo in EVs with high CAF-programming activity. Mechanistically, EV-associated SPP1 mainly interacted with CD44 on fibroblasts to activate PI3K–AKT signaling, leading to sustained differentiation into the MMP11⁺ CAF phenotype. Clinically, the presence of MMP11⁺ CAFs was associated with dense matrix buildup, immune exclusion, reduced intratumoral T-cell infiltration, and poor responses to chemo-immunotherapy. Disrupting the EV–SPP1 pathway reduced MMP11⁺ CAF development, attenuated stromal structure, revived anti-tumor T-cell immunity, and increased tumor sensitivity to PD-1 blockade in vivo. These findings define MMP11⁺ CAFs as a functionally pro-tumorigenic fibroblast state selectively programmed by tumor-derived EV-associated SPP1, providing mechanistic evidence linking stromal reprogramming to immune exclusion and immunotherapy resistance.
The classical abscopal effect is defined here as objective regression of a verified non-irradiated tumor lesion following local radiotherapy. An immune abscopal phenomenon denotes a prespecified, reproducible immune change within such a lesion that is attributable to the local intervention, with or without radiographic regression. Systemic immune reprogramming is a broader, direction-neutral term for reproducible longitudinal changes in immune composition, clonality, activation, trafficking, or suppression across peripheral blood and/or multiple lesions after local therapy; blood-only changes do not constitute an immune abscopal phenomenon or establish clinical benefit. Using these non-interchangeable definitions, we examine local immunogenic injury, dendritic-cell cross-presentation, T-cell expansion and trafficking, distant-tumor permissiveness, organ-niche restraint, and unfavorable systemic trajectories, including treatment-induced distant suppression. Mechanistic causality is supported mainly by preclinical studies, whereas human evidence derives from observational studies, correlative analyses embedded in clinical trials, and heterogeneous interventional trials that report occasional distant responses without establishing reproducible abscopal efficacy. Established radiographic and metabolic response criteria are therefore separated from the author-proposed Grade A/B/C framework, five-domain modality comparison, tiered monitoring strategy, and investigational adaptive loop, all of which require prospective validation. This graphical abstract illustrates immunogenic tumor injury triggered by local therapies including radiotherapy, HIFU, histotripsy and sonodynamic therapy. It contrasts favorable abscopal immune cascades with restrained immune remodeling driven by Tregs, MDSCs, M2 macrophages, IL-10 and TGF-β. Key assessment pillars for authentic abscopal effect include imaging, peripheral immune readouts, distant tumor microenvironment profiling and confounder exclusion.
Drug-tolerant persister (DTP) cells survive anticancer therapy through reversible adaptive states and can contribute to tumor regrowth. However, how colorectal cancer (CRC) DTP cells preserve mitochondrial fitness during chemotherapy remains unclear. We investigated the metabolic and post-translational mechanisms underlying the FOLFOXIRI-induced DTP state. FOLFOXIRI-induced DTP–regrowth models were established using CRC patient-derived xenografts, PDX-derived organoids, cell lines, and cell line-derived xenografts. Whole-exome sequencing and transcriptomic, proteomic, metabolomic, and lactylomic analyses (global mass-spectrometry-based profiling of lysine lactylation sites) were integrated with genetic and pharmacological perturbations, metabolic and mitochondrial assays, protein-stability and ubiquitination analyses, and a cell-free delactylation assay, assessing the enzymatic removal of lysine lactylation. Clinical relevance was evaluated in chemotherapy-exposed CRC tissues and circulating tumor cells (CTCs). FOLFOXIRI-surviving CRC cells exhibited reduced proliferation without increased apoptosis, resumed growth after drug withdrawal, retained FOLFOXIRI sensitivity upon regrowth, and showed reversible molecular and metabolic remodeling. DTP cells displayed suppressed glycolytic activity, reduced lactate production, and increased reliance on oxidative phosphorylation, accompanied by activation of mitophagy. The mitochondrial complex III subunit UQCRC2 accumulated in DTP cells and supported Parkin (PRKN)/SQSTM1-associated mitophagy and residual-cell survival. Reduced lactate production was accompanied by decreased global lysine lactylation and reduced UQCRC2 lactylation at lysine 430 (UQCRC2-K430la). Increased UQCRC2-K430la promoted K48-linked ubiquitination and proteasome-dependent degradation of UQCRC2. SIRT1 directly delactylated UQCRC2 at K430 in an NAD+-dependent manner, thereby reducing K48-linked ubiquitination and stabilizing UQCRC2. Depletion of PINK1 or UQCRC2 and genetic or pharmacological inhibition of SIRT1 reduced residual DTP-cell survival and delayed regrowth. Among chemotherapy-responsive patients with advanced CRC, low tumor UQCRC2-K430la expression remained associated with poorer overall survival after multivariable adjustment. In paired CTC samples, increased SIRT1 or decreased UQCRC2-K430la expression after chemotherapy was associated with shorter progression-free survival. These findings identify a low-lactate/SIRT1–UQCRC2-K430la mechanism that couples glycolytic suppression to mitophagy-associated mitochondrial quality control and enables reversible chemotherapy tolerance in CRC. This pathway represents a candidate therapeutic vulnerability and supports further evaluation of UQCRC2-K430la as a biomarker of chemotherapy-associated residual disease.
