Pancreatic ductal adenocarcinoma (PDAC) develops within a biomechanically abnormal tumor microenvironment, characterized by a dense stroma and elevated compressive forces. While extracellular matrix stiffness has been extensively studied, the impact of compressive forces on immune regulation and tumor–immune interactions remains poorly understood. We integrated two complementary bioengineered compression models, a 2D transmembrane pressure device and confined 3D spheroids, with bulk transcriptomic and Liquid Chromatography–Mass Spectrometry (LC-MS)–based exometabolomic profiling to examine how mechanical compression shapes macrophage behaviour and tumor–immune crosstalk. Controlled compressive stress (0–8 mmHg) was applied to macrophages, tumor cells, and tumor–macrophage cocultures, followed by pathway analysis, functional assays, and multi-omic integration. Mechanical compression activated conserved mechanotransduction pathways in macrophages, including PI3K/Akt and MAPK/SAPK signaling, and induced transcriptional programs associated with inflammatory activation consistent with an M1-like macrophage phenotype and cytoskeletal remodelling. In parallel, compressed tumor cells adopted an immunomodulatory state marked by increased expression of immunosuppressive cytokines and macrophage checkpoint signals. When tumor cells and macrophages were simultaneously exposed to compression, functional assays revealed a shift of macrophages toward immunosuppressive phenotypes. Bulk RNA sequencing identified cell-type–specific transcriptional responses converging on metabolic pathways, while LC-MS exometabolomics revealed compression-dependent enrichment of extracellular nucleotide metabolites in tumor–macrophage cocultures. These findings identify compressive stress as a critical regulator of immune suppression and tumor–immune metabolic coupling in PDAC, highlighting mechanical forces as important drivers of immune dysfunction in mechanically constrained tumors.

Adoptive T cell therapies have transformed the treatment of selected haematological malignancies but remain limited in many cancer settings by biological barriers that restrict effective and durable antitumour responses. Here, we investigated whether pharmacological blockade of the endothelin receptor pathway, which regulates vascular, stromal and immune processes relevant to antitumour responses, could enhance T cell-based cellular immunotherapy. Using orthotopic 4T1 triple-negative breast cancer and A20 B cell lymphoma models, we evaluated endothelin receptor blockade with non-engineered adoptive T cell therapy and anti-CD19 CAR T cell therapy, respectively, with or without immune checkpoint inhibition. Endothelin receptor blockade substantially increased objective responses across both therapeutic platforms, while the addition of immune checkpoint inhibition further increased response frequency and promoted maintained complete responses, resulting in the most durable tumour control. Mice achieving maintained complete responses resisted tumour rechallenge, consistent with long-term antitumour immune protection. High-dimensional T cell profiling identified distinct intratumoral T cell states associated with tumour control and elevated CD2 expression as a recurring feature across these populations. Together, these findings identify endothelin receptor blockade as a rational combinatorial strategy for enhancing T cell-based cellular immunotherapy and provide a strong rationale for the clinical evaluation of this therapeutic strategy.

Immunotherapy has transformed cancer treatment but remains ineffective in many solid tumors, largely due to the immunosuppressive tumor microenvironment (TME). A dense/stiff extracellular matrix (ECM) can hinder immune cell infiltration, limiting therapeutic success. To overcome this challenge, we developed a live biotherapeutic platform using engineered programmable bacteria that offer advantages for delivering enzymes that normalize the TME. These nonpathogenic, commensal-derived strains are equipped with tumor-inducible promoters to deliver hyaluronidase locally and safely within the TME. Our study demonstrates that bacterial-mediated hyaluronan (HA) degradation reduces tumor stiffness, restores vascular function, and enhances immune checkpoint inhibitor efficacy in breast cancer murine models by improving immune cell infiltration and activation. Using machine learning and feature importance analysis, we identified biomarkers predictive of therapeutic response. Notably, we found that the potency of antitumor responses depends highly on baseline stiffness levels and ECM composition in relatively stiff breast tumors. In contrast, responses in less stiff colorectal cancer models characterized by low HA deposition primarily associated with immune cell composition, particularly CD8+ and CD4+ T cells and M2 macrophages. Our study showcases the potential of programmable bacteria to reshape the TME and identifies distinct tumor-dependent mechanisms of response across different tumor types, paving the way for more effective cancer treatments.

Nano-immunotherapy shows great promise in improving patient outcomes, as seen in advanced triple-negative breast cancer, but it does not cure the disease, with median survival under two years. Therefore, understanding resistance mechanisms and developing strategies to enhance its effectiveness in breast cancer is crucial. A key resistance mechanism is the pronounced desmoplasia in the tumor microenvironment, which leads to dysfunction of tumor blood vessels and thus, to hypoperfusion, limited drug delivery and hypoxia. Ultrasound sonopermeation and agents that normalize the tumor stroma have been employed separately to restore vascular abnormalities in tumors with some success. Here, we performed in vivo studies in two murine, orthotopic breast tumor models to explore if combination of ultrasound sonopermeation with a stroma normalization drug can synergistically improve tumor perfusion and enhance the efficacy of nano-immunotherapy. We found that the proposed combinatorial treatment can drastically reduce primary tumor growth and in many cases tumors were no longer measurable. Overall survival studies showed that all mice that received the combination treatment survived and rechallenge experiments revealed that the survivors obtained immunological memory. Employing ultrasound elastography and contrast enhanced ultrasound along with proteomics analysis, flow cytometry and immunofluorescene staining, we found the combinatorial treatment reduced tumor stiffness to normal levels, restoring tumor perfusion and oxygenation. Furthermore, it increased infiltration and activity of immune cells and altered the levels of immunosupportive chemokines. Finally, using machine learning analysis, we identified that tumor stiffness, CD8+ T cells and M2-type macrophages were strong predictors of treatment response.

Ketotifen alleviates hypoxia and restores intratumoral T cell infiltration by upregulating immune cell adhesion to blood vessels.

Ketotifen mechanotherapeutic enhances the neoadjuvant treatment by reducing the recruitment of immunosuppressive cell populations.