Background: In critical illness, clinical status evolves at different rates and patients may present at varying lengths of time since disease onset. If treatment effects are time dependent, characterizing temporal heterogeneity of patient cohorts may enable enrichment for larger effect sizes and may result in improved treatment protocols for individual patients. Methods: Using daily clinical and laboratory values from patients undergoing mechanical ventilation at 9 ICUs across 6 centers in Toronto, Canada, we first perform a latent class analysis and identify patients who experience transitions among classes. We next construct time autocorrelation functions of individual clinical variables, including class defining variables, and propose the auto-correlation time as an estimate of time since onset of illness and of susceptibility to therapeutic intervention. Results: We find that the data is best described by two latent classes with different severity of illness and mortality. Out of 14,196 patients in our cohort, 1,730 were switchers who made at least one transition between classes, whereas 12,466 were non-switchers. Most transitions occurred in the first three days of the ICU stay. The distribution of clinical variable auto-correlation times was asymmetric, peaking at short times but with a long tail consistent with heterogenous dynamics. We further find that autocorrelation time distributions exhibit signs of aging, with the distributions shifted towards longer times later in the ICU stay. Interpretation: In this cohort of patients with respiratory failure we found distinct temporal profiles with some patients transitioning between latent classes and some patients characterized by adynamic trajectories. Dynamics generally slow with time since presentation, suggesting that dynamic scores may be a proxy for time since onset of illness. At any point in time, however, quantitative measures of dynamics such as the autocorrelation time are broadly distributed, suggesting dynamics remain heterogenous throughout the ICU course. Measures of dynamic stability may be useful both for identifying points in time where treatment effect may be expected to be maximal (prior to the onset of adynamic behavior) and for assembling temporally homogeneous trial cohorts for both prognostic and predictive enrichment.

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, with a 5-year overall survival of ∼13%. Delayed diagnosis, limited response to current treatments, and the predominance of locally advanced or metastatic disease contribute to poor outcomes. Leveraging our preclinical findings in PDAC murine models, we showed that adding losartan—an angiotensin II type-1 receptor (AT1) blocker—to FOLFIRINOX followed by chemoradiation doubled R0 resection rates to ∼70% in locally advanced PDAC in a phase II trial (NCT01821729) (PMID: 31145418). However, the patient-response was variable. Here we recapitulated this variability in orthotopic PDAC mouse models and revealed the underlying mechanism. Methods and Results: To investigate potential causes of the variable response, we mapped AT1 expression using light-sheet microscopy in AT1 reporter mice bearing orthotopic PDAC. We observed abundant AT1 throughout the tumor microenvironment. To define its functional relevance, we generated Agtr1a knockout (KO) PDAC cell lines and inducible KO mouse models. AT1 deletion in either cancer cells or stromal cells (but not α-SMA+ myCAFs or pericytes) significantly reduced tumor growth. Because losartan is a pro-drug that needs activation by liver enzymes CYP3A4 and CYP2C9, we next examined its metabolism. We subcutaneously administered two major losartan metabolites, EXP3179 and EXP3174, to orthotopic PDAC-bearing mice and found that EXP3174 mediated the anti-tumor effects of losartan. Mass spectrometry of plasma samples revealed that while some tumor-bearing mice efficiently converted losartan to its active metabolite, others showed limited or no conversion. To determine the cause, we assessed CYP2C9 and CYP3A4 activity in liver microsomes from non-metastatic PDAC-bearing mice. CYP2C9 activity was markedly reduced and inversely correlated with tumor burden, providing a mechanistic basis for differential losartan activation. Ongoing work aims to determine whether PDAC patients likewise display variable plasma levels of losartan and EXP3174. Conclusion: These findings identify impaired hepatic metabolism as a key driver of variable losartan responses in locally advanced PDAC. Mass-spectrometry-based assessment of losartan and EXP3174 levels may help determine which patients can effectively activate the drug. Given losartan’s safety and low cost, its oral administration remains appropriate for most patients; however, in individuals with compromised CYP2C9 activity, administration of the active metabolite — EXP3174 may represent a more effective therapeutic option. Citation Format: Heena Kumra, Ryo Morisue, Benjamin E. Wolf, Vasiliki Salameti, Sonu Subudhi, Nilesh P. Talele, Eric F. Zaniewski, Robert Morris, Tsion H. Tale, Karim El-Marouk, Cora Schueller, Mariagiovanna Barresi, Jennifer Schulz, Halil I. Corbali, Rieke Schleinhege, Peigen Huang, Pascal Bernatchez, Wilhelm Haas, Yves Boucher, Dai Fukumura, Rakesh K. Jain. Improving response of pancreatic cancer to losartan: Mechanistic insights and implications for personalized therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1781.

Cancer cell proliferation requires a precise balance between biomass production and nutrient catabolism. The pyridine nucleotide cofactors nicotinamide adenine dinucleotide NAD(H) and NAD phosphate NADP(H) are central to this process, but their compartment-specific regulation is incompletely understood. Using in vivo isotope-labeled metabolite tracing in an orthotopic xenograft model, we find that human gliomas extensively synthesize proline, an amino acid previously associated with hypoxia tolerance. In glioma cells, we identify a hypoxia-enhanced proliferative sensitivity to environmental proline dependent on NADH to NADPH transhydrogenation from a spatially compartmentalized mitochondrial pool by the enzyme nicotinamide nucleotide transhydrogenase (NNT). We demonstrate NNT-dependent generation of mitochondrial NADPH is important for proline accumulation, maintenance of antioxidant systems, and reductive metabolism in hypoxic glioma cells in vitro and tumor progression in vivo. Collectively, these results highlight proline accumulation as a marker of mitochondrial NAD(P)(H) homeostasis and NNT as a specific metabolic dependency in human glioma.

Abstract Introduction: Chronic arterial hypertension (CAH) is one of the most prevalent medical conditions worldwide and plays a causal role in cardiovascular diseases. Cancer patients with CAH have inferior outcomes compared to non-hypertensive cancer patients, especially in pancreatic ductal adenocarcinoma (PDAC). Furthermore, retrospective analyses have shown that antihypertensive therapy targeting angiotensin signaling is associated with improved oncological outcomes compared to other anti-hypertensive treatments in cancer patients. Methods and Results: To investigate this relationship, we employed a syngeneic, orthotopic PDAC model in mice with chronic hypertension induced by subcutaneously implanted osmotic minipumps delivering angiotensin II. Using highly multiplexed spectral flow cytometry, we found that chronic angiotensin II administration re-wires the anti-tumor immune response, leading to an increased presence of myeloid cells, particularly Ly6C+ monocytes, and CD206+ macrophages. Blockage of the angiotensin II receptor type I (AT1) with losartan leads to a marked decrease in tumor growth and a reduction in MDSCs. This effect was observed only with the synchronous administration of angiotensin II and losartan, not with losartan alone, and was consistent across different PDAC treatment regimens. In addition, we observed a trend toward improved tumor vessel perfusion in hypertensive mice treated with losartan, indicating vascular repair. Additional findings suggest that inhibition of the angiotensin II receptor type 2 (AT2) and the Mas receptor (which physiologically counterbalance AT1 signaling) abrogated the beneficial effect of AT1 blockade with losartan, suggesting a role for AT2 and the Mas receptor in shaping the immune microenvironment. Conclusion: Overall, our findings show that CAH reprograms the tumor microenvironment in PDAC and that inhibition of angiotensin signaling reverses these changes. This may indicate that inhibition of angiotensin signaling in cancer patients with CAH may exert its beneficial effect by reprogramming the anti-tumor immune response. Citation Format: Benjamin Wolf, Heena Kumra, Ryo Morisue, Karim El-marouk, Igor L. Gomes-Santos, Rieke Schleinhege, Tsion H. Tale, Marc Charabati, Sonu Subudhi, Dai Fukumura, Rakesh K. Jain. Angiotensin-II-induced chronic hypertension reprograms the antitumor-immune-response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2084.

Non-NF2 Schwannomatosis (SWN) is a genetic disorder characterized by multiple non-malignant schwannomas growing on the spine and peripheral nerves. Patients with SWN overwhelmingly present with intractable chronic pain. There are no FDA-approved drugs to halt tumor growth or alleviate pain. Research on SWN is hindered by the lack of clinically relevant models. We established patient-derived SWN cell lines from patients with varying pain levels and developed orthotopic patient-derived xenograft models that reproduce patients' pain responses. We further developed a novel dorsal root ganglia (DRG) imaging model for longitudinal intravital imaging of macrophage infiltration into the DRG and sensory neuron pain response. Leveraging these novel models, we found that Schwannomas grown distantly in the peripheral nerve caused an influx of macrophages into the DRG. These macrophages in the DRG caused pain via overproducing IL-6. Treatment with anti-IL-6 antibody reduced pain but had modest efficacy in tumor control. We identified epidermal growth factor receptor (EGFR) signaling as a key driver of schwannoma growth and an escape mechanism from anti-IL6 treatment. Finally, we found that combining IL-6 and EGFR blockade effectively controlled pain and tumor growth simultaneously in SWN models. In summary, we elucidated the cellular and molecular crosstalk between schwannoma (HMGB1), neuron (CCL2), and macrophage (IL-6) in driving pain, and identified the EGF signaling pathway as a driver of SWN tumor progression, thereby uncovering novel therapeutic targets that may improve clinical management of SWN.

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. Here we quantify the absolute levels of 124 metabolites in multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different tissue sites. We then asked how tumour growth in different tissues relates to nutrient availability and tumour biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. In addition, we identify purine synthesis as a requirement for tumour growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumour de novo nucleotide synthesis activity. These data suggest that a complex interplay between multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

Venn plots and selected GSEA pathways.

Relationship between pathological response and circulating biomarker levels in the plasma of treated patients.

Losartan+FFX+CRT and FFX+CRT gene correlation with overall survival (OS).

Longitudinal analysis of circulating biomarkers in the plasma of treated patients.

Immunofluorescence staining of residual PDAC in pathological responders and non-responders in FFX+CRT and losartan+FFX+CRT.

Heatmap showing differentially expressed genes (DEGs) and their expression in each patient in FFX+CRT and losartan+FFX+CRT-treated groups.

Group 3 Medulloblastoma (G3MB) is a pediatric brain cancer with poor prognosis, and a median 5-year overall survival of 45-58% in infants and young children, respectively. Moreover, the current standard-of-care therapy – which includes spinal radiation – causes tremendous morbidity. While immunotherapy with immune checkpoint blockade has transformed the treatment of many malignancies, it has yet to show benefit in the most common form pediatric brain tumors. Here we show that B cell depletion using Rituximab; anti-CD20: -- an FDA-approved antibody -- enhances anti-tumor immunity, promote tumor regression and improves overall survival of mice bearing orthotopic murine models of G3MB. By analyzing of human MB transcriptomics datasets, we discovered that pediatric MB patients with elevated B cell signatures have significantly reduced survival rates compared to signatures of other immune cell populations. Using immunohistochemistry and transcriptomic analysis of orthotopic murine models of G3MB, we found that intratumoral B cells are immunosuppressive – they directly inhibit anti-tumor CD8 T cells and reprogram the tumor microenvironment to an immunosuppressive milieu. Furthermore, systemic B cell depletion using murine equivalents of the FDA approved B cell depleting antibody (Rituximab; anti-CD20: 100µg/dose) significantly enhanced animal survival, and improved T cell infiltration and activation – estimated by flow cytometry and single cell RNA-sequencing. Furthermore, systemic B cell depletion enhanced the therapeutic efficacy of murine equivalents of standard-of-care radiation therapy (20.7Gy to tumor bed) and synergized with fractionated low-dose radiation therapy (1.81Gy x 6 daily doses) – which is likely to be less toxic to the developing pediatric brain. Finally, our mechanistic studies done revealed that tumor - derived placental growth factor (PlGF) reprograms B cells toward an immunosuppressive phenotype, characterized by increased IL-10 expression and suppression of CD8+ T cell function. These findings also suggest a potential benefit of anti-PlGF antibody treatment of MB in patient-derived models and its safe use in patients (Snuderl et al, Cell 2013; Sulnier-Sholler et al. Clinical Cancer Research 2022). In conclusion, our findings suggest that targeting B cells systemically represents a promising therapeutic strategy for G3MB treatment while preserving patients’ quality of life. The availability of FDA-approved B cell-targeting drugs for pediatric malignancies offers a path for rapid clinical translation, particularly crucial for this devastating disease. Citation Format: Ashwin S Kumar, Taylor P Uccello, Igor L Gomes dos Santos, Vasiliki Salameti, Sophie C Steinbuch, Sonu Subudhi, Rakesh K Jain. Targeting B Lymphocytes to Improve Therapeutic Outcomes for Pediatric Medulloblastoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B095.

Quantitative analysis of immunofluorescence staining in PDAC lesions from FFX+CRT-treated patients.

Top 30 upregulated and 30 downregulated genes in treatment group versus untreated.

Immune checkpoint blockade therapy, including anti-PD-1 antibody (αPD1), has shown limited success in glioblastoma (GBM), a lethal brain tumor with a median survival of under two years. The Wnt signaling pathway - known for its role in development - can limit treatment outcomes in several malignancies, including GBM. Moreover, it plays a critical role in GBM progression by promoting stemness and epithelial-mesenchymal transition. While the preclinical evidence indicates that Wnt signaling is linked to anti-PD-1 (αPD1) resistance in extracranial tumors, its role in GBM immune suppression and αPD1 resistance remains unknown. Here, we show that Wnt7b is upregulated in GBM patients and contributes to αPD1 therapy resistance in a clinically relevant, stemness-rich murine GBM syngeneic model (GSC005). We observed that elevated Wnt7b and β-catenin levels correlated with a poor αPD1 response. We hypothesized that inhibiting Wnt signaling could sensitize tumors to αPD1 therapy by reprogramming the TME from immunosuppressive to immunostimulatory and bolster anti-tumor immunity. We orthotopically implanted GSC005 into immunocompetent mice, then tracked tumor growth using ultrasound, and analyzed tumor and immune cell subpopulations by flow cytometry. Combining the porcupine inhibitor WNT974 with αPD1 improved median survival from 25 to 59 Days (p < 0.05), with 25% of mice achieving long-term survival. WNT974 with αPD1 increased DC3-like dendritic cells and reduced granulocytic myeloid-derived suppressor cells (gMDSCs). Moreover, this combination increased the Ki67+ CD8 T/Ki67+ Treg ratio, shifting the balance towards an anti-tumor immune response and upregulated Gzmb in CD8 T cells, enhancing their effector function. Interestingly, resistance to WNT974 + αPD1 was marked by elevated monocytic MDSCs, pERK/MAPK pathway activation, and modest T cell exhaustion. Our study provides compelling data and rationale to repurpose WNT974, reported to be safe in a Phase I trial in extracranial cancer patients, in combination with αPD1 therapy in GBM patients with elevated Wnt7b/β-catenin signaling.

Quantitative analysis of immunofluorescence staining in PDAC lesions from FFX+CRT and losartan+FFX+CRT-treated groups.

Differentially expressed genes (DEG) in losartan+FFX+CRT versus FFX+CRT, losartan+FFX+CRT versus untreated, and FFX+CRT versus untreated.

Gene sets associated with overall survival in losartan+FFX+CRT and FFX+CRT-treated groups.