Faculty Bibliography

Nerves and cancer-associated fibroblasts (CAFs) have each been shown to regulate cancer progression directly. However, whether these cells interact to control tumor progression remains largely unknown. We show that in colorectal cancer (CRC), cholinergic stimulation induces CHRM3/Gq-dependent NTN1 secretion from CAFs, which in turn enhances intratumor cholinergic innervation. Within this feedforward loop, cholinergic stimulation promotes CRC growth directly through tumoral CHRM3/Gq-mediated YAP activation, while CAF-derived NTN1 promotes CRC growth and epithelial-to-mesenchymal transition-like programs via UNC5B-PI3K/AKT signaling. Chemogenetic activation of cholinergic neurons or fibroblast activation of the M3 receptor/Gq promotes tumoral YAP and AKT signaling and CRC progression. Conversely, blocking CHRM3 or NTN1 suppresses these pathways and improves mouse survival. In human CRC, high NTN1 expression is associated with a mesenchymal-like subtype and poor patient outcomes. These findings suggest that the neuro-mesenchymal interaction is central to CRC progression and could be therapeutically targeted with a CHRM3 antagonist or NTN1-blocking antibody.

BACKGROUND:Neural regulation contributes to pancreatic ductal adenocarcinoma (PDAC) development but effects of neural-targeting medications on presentation or outcomes remain unclear. METHODS:We conducted a retrospective study using the Veterans Affairs (VA) Corporate Data Warehouse (CDW) to identify patients with pancreatic cancer (2000-2020). Exposure to beta blocker, cholinergics or statins was defined by active prescriptions within 6 months before or 1 month after diagnosis. Outcomes included histologic subtype, stage, and overall survival (OS). Propensity score matching was performed for each medication class, with survival assessed using Kaplan-Meier and Cox regression models. RESULTS:Among 7,578 Veterans with pancreatic cancer, 76% had adenocarcinoma and 60% presented with stage IV disease. Beta blocker use was associated with lower odds of advanced stage (OR 0.55, 95% CI 0.5-0.63, p<0.0001) and improved OS (HR 0.89, 95%CI 0.84-0.95, p<0.0001). Cholinergic agonist use was associated with reduced likelihood of adenocarcinoma histology (OR 0.64, 95% CI 0.41-1.01, p=0.051) and advanced stage (OR 0.53, 95%CI 0.34-0.85, p=<0.0001), but not OS. Statin use was associated with adenocarcinoma histology (OR 1.24, 95%CI 1.09-1.43, p=0.002) and lower odds of advanced stage (OR 0.77, 95%CI 0.68-0.88, p=<0.0001). CONCLUSIONS:Beta blockers were associated with earlier stage and improved survival. Cholinergic agonists and statins were associated with earlier stage. Cholinergic agonists were linked to lower likelihood of adenocarcinoma histology, while statins to higher likelihood. These findings suggest neural and metabolic pathways may shape early PDAC biology. IMPACT/CONCLUSIONS:Autonomic and metabolic pathways may influence PDAC biology. Beta blockers merit mechanistic and clinical evaluation as adjunctive therapies.

BACKGROUND:Epigenetic regulators represent a novel strategy to modulate the tumor immune microenvironment in pancreatic ductal adenocarcinoma (PDAC). In preclinical models, DNA hypomethylating agents enhance cytotoxic T-cell infiltration, synergize with PD-1 blockade, and improve survival when combined with immune checkpoint blockade. This single-institution, phase II study evaluated the safety, efficacy, and biomarkers of azacitidine plus pembrolizumab in patients with previously treated PDAC. METHODS:Patients with locally advanced or metastatic PDAC after one prior regimen received 50 mg/m2 subcutaneous azacitidine on days 1-5 of a 28-day cycle, starting week 1, and pembrolizumab 200 mg intravenously every 3 weeks starting week 3. Baseline and on-treatment blood and tumor was collected for exploratory biomarker analysis. RESULTS:Thirty-six patients enrolled between October 2017 and September 2021 (median age: 62.5 years); 34 were evaluable for safety; 31 for efficacy. Treatment was generally well-tolerated, with Grade 1-2 fatigue and diarrhea most common AEs. Three patients (9.7%) had a partial response, and the disease control rate was 35.5%. Median progression-free and overall survival was 1.51 and 4.83 months, respectively. Exploratory analysis suggested higher baseline CD8+ T cells and lower tumor Ki-67 was associated with response, whereas low baseline CD8+ T cell and Granzyme B infiltration correlated with higher exponential tumor growth rate. PD-L1 and CD68 expression were not predictive of benefit. CONCLUSION/CONCLUSIONS:Azacitidine plus pembrolizumab demonstrated limited clinical activity in second line, locally advanced or metastatic PDAC. Biomarker analysis suggests higher baseline CD8+ T-cell infiltration and lower proliferative index may identify patients more likely to benefit. (NCT03264404).

Nerves can regulate tumorigenesis and cancer progression. However, clarification of the role of axon guidance molecules in tumorigenesis, innervation, and metastasis is required to better understand the tumor-promoting functions of nerves. Using murine KrasG12D-mutant pancreatic organoids, we screened axon guidance molecules and identified netrin-1 upregulation. Netrin-1 was also upregulated in vivo during pancreatic tumorigenesis in humans and mice. Mutant KRAS and β-adrenergic signaling upregulated netrin-1 and its receptor NEO1 in epithelial cells in part through the MAPK pathway. Ex vivo culture of celiac ganglia showed that netrin-1 promoted the axonogenesis of sympathetic neurons through nerve NEO1. In the Pdx1-Cre;LSL-KrasG12D/+ model, knockout of Ntn1, which encodes netrin-1, decreased sympathetic innervation and the development of pancreatic intraepithelial neoplasia. Treatment of pancreatic tumor organoids with recombinant netrin-1 enhanced cell growth, epithelial-mesenchymal transition (EMT), and cancer stemness with the upregulation of ZEB1 and SOX9 through NEO1-mediated activation of focal adhesion kinase (FAK). In Pdx1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ mice, Ntn1 knockout reduced innervation, FAK phosphorylation, and the features of EMT and stemness to extend mouse survival. In a liver metastasis model of pancreatic ductal adenocarcinoma (PDAC), treatment with a netrin-1-neutralizing antibody or tumoral knockout of Neo1 reduced ZEB1 and SOX9 and decreased tumor progression. In contrast, netrin-1 overexpression promoted innervation and the progression of PDAC liver metastasis. These data suggest that the netrin-1/NEO1 axis is a key regulator of PDAC progression, directly influencing cancer cell stemness and EMT, while indirectly promoting tumor growth through nerves. Inhibiting the netrin-1 pathway could represent a potential therapeutic approach for PDAC.

INTRODUCTION/UNASSIGNED:Lymphoepithelioma-like carcinomas (LELC) are rare tumors composed of poorly differentiated epithelial cells with a prominent lymphoid stromal infiltrate. This tumor histology has been described in various tissues of origin including the nasopharynx, lung, thymus, breast, bladder, and others. However, involvement of the gastrointestinal system is relatively uncommon and has most often been described in the stomach. CASE PRESENTATION/UNASSIGNED:Here, we report a case of a young woman who presented with metastatic disease of unclear primary involving the liver and abdominal lymph nodes, along with marked Epstein-Barr virus (EBV) viremia. She was ultimately found to have metastatic EBV-associated lymphoepithelioma-like carcinoma of likely small bowel origin, an uncommon tissue of origin for a rare histopathology. She was treated with chemotherapy and immunotherapy regimens commonly used for gastrointestinal and biliary cancers, with an initial treatment response but ultimately rapid tumor progression. CONCLUSION/UNASSIGNED:LELCs are rare tumors that uncommonly involve the gastrointestinal system, particularly the small bowel. Further reports are needed to better understand disease presentation, pathophysiology, prognosis, and management.

Pathologically activated immunosuppressive neutrophils impair cancer immunotherapy efficacy. The chemokine receptor CXCR4, a central regulator of hematopoiesis and neutrophil biology, represents an attractive target. Here, we fuse a secreted CXCR4 partial agonist, trefoil factor 2 (TFF2), to mouse serum albumin (MSA) and demonstrate that TFF2-MSA peptide synergizes with anti-PD-1 to inhibit primary tumor growth and distant metastases and prolongs survival in gastric cancer (GC) mouse models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) in vivo, we find that TFF2-MSA selectively reduces the Hdc-GFP⁺CXCR4^high immunosuppressive neutrophils, thereby boosting CD8⁺ T cell-mediated tumor killing with anti-PD-1. Importantly, TFF2-MSA reduces bone marrow granulopoiesis, contrasting with CXCR4 antagonism, which fails to confer therapeutic benefits. In GC patients, elevated CXCR4⁺LOX-1⁺ low-density neutrophils correlate with lower circulating TFF2 levels. Collectively, our studies introduce a strategy that utilizes CXCR4 partial agonism to restore anti-PD-1 sensitivity by targeting immunosuppressive neutrophils and granulopoiesis.

The bone marrow (BM) is altered in obesity to promote myeloid cell generation, but the mechanisms driving these changes remain unclear. Here, we show that obesogenic stimuli promote adipose tissue macrophages to recruit neutrophils from the BM in mice. Recruitment of BM neutrophils activates hematopoietic stem cells, which produce myeloid cells that accumulate in the circulation and drive inflammation. This recruitment is not resolved by weight loss, leading to sustained myelopoiesis in previously obese mice. Inhibiting neutrophil recruitment out of the BM in obese mice or during weight loss reduces BM myelopoiesis and adipose tissue inflammation, and improves glucose tolerance. In humans with obesity, plasma neutrophil chemokines are increased, correlate with increased insulin resistance, but do not decrease with weight loss. Our results demonstrate that neutrophil recruitment is a key mediator of myelopoiesis during obesity, and targeting this pathway is a potential strategy to improve inflammation during obesity and weight loss.

BACKGROUND:The parasympathetic branch of the autonomic nervous system has shown tumor-suppressive effects in preclinical models of pancreatic adenocarcinoma (PDAC) by inhibiting cancer stem cells and suppressing inflammatory cytokine production. Based on these findings, we hypothesized that bethanechol, an FDA-approved parasympathomimetic agent targeting muscarinic receptors, could enhance treatment efficacy in PDAC. METHODS:We conducted a Phase 0/window of opportunity study evaluating short term parasympathetic activation with fixed dose bethanechol (100 mg twice daily) in subjects with resectable or borderline resectable PDAC prior to surgery. The primary endpoint was change in cell proliferation by Ki-67 expression compared to stage matched controls. Secondary endpoints included tissue expression of stem cell markers (CD44), infiltrating immune cells (CD8a, Granzyme B, and CD68), and changes in circulating inflammatory cytokine concentrations. RESULTS:Seventeen patients were enrolled with 13 eligible for analysis of endpoints. Median age was 74 (59-86), 6 female (46%), all ECOG 0-1 and median duration of treatment was 8 days (7-13). R0 resections were achieved in 9 patients (69%). There was no difference in Ki67 and CD44 tissue biomarkers between bethanechol-treated and control samples. Decreased numbers of Granzyme B-expressing cells were seen in bethanechol-treated tissues. Bethanechol treatment was associated with suppression of circulating IL-18. The most common treatment related adverse events (TRAE) were hot flashes (30.7%), urinary frequency (15.4%), increased salivation (15.4%), hyperhidrosis (7.7%), and nausea (7.7%). There were no Grade 3 or higher adverse effects. No surgical complications were attributed to bethanechol treatment. CONCLUSION/CONCLUSIONS:Bethanechol 100 mg twice daily is well tolerated in patients with PDAC in this small phase 0/window of opportunity study (NCT03572283). Bethanechol treatment was associated with decreased Granzyme B positive cells and decreased circulating IL-18 consistent with an anti-inflammatory role for parasympathetic muscarinic signaling in PDAC.

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

BACKGROUND:Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS:We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS/RESULTS:Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS:Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING/BACKGROUND:1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).