Frankel, Timothy L.

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer, is a deadly disease with a complex tumor microenvironment (TME). Cytotoxic combination chemotherapy treatments are the mainstay of PDAC therapy, but patients rapidly develop chemoresistance, highlighting the need to elucidate how chemotherapy alters the TME and whether these changes drive chemoresistance. Here, we examined matched pre- and post-treatment tissue specimens using single-cell RNA-sequencing and found near-universal enrichment after treatment of axonal guidance genes in cancer-associated fibroblasts (CAFs). These neural CAFs were enriched near sites of perineural invasion, coinciding with regions of increased tumor cell proliferation, and were enriched in tumor areas distant from nerves after chemotherapy. Metastatic recurrent lesions had the highest prevalence of neural CAFs versus primary tumors and untreated metastases. Neural CAFs showed elevated non-canonical WNT mediators and axonal guidance genes, which complemented matching cognate binding partners in tumor epithelial cells, suggesting a role in tumor-stroma crosstalk. Additionally, in vitro studies revealed enrichment of axonal guidance genes in patient-derived CAF lines upon exposure to chemotherapy or tumor cells. Altogether, these findings implicate fibroblast-derived axonal-guidance genes in promoting PDAC invasion and point to a promising therapeutic target for this disease.

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer, is a deadly disease with a complex tumor microenvironment (TME). How chemotherapy alters the TME, and whether these changes drive chemoresistance, is poorly understood. We examined matched pre- and post-treatment tissue specimens and found near-universal enrichment of axonal guidance genes in cancer-associated fibroblasts (CAFs) after treatment. These CAFs were enriched near sites of perineural invasion, coinciding with regions of increased tumor cell proliferation, and were enriched in tumor areas distant from nerves after chemotherapy. Metastatic recurrence lesions had the highest prevalence of these CAFs versus primary tumors and untreated metastasis. These CAFs showed elevated non-canonical Wnt mediators and axonal guidance genes, which complemented matching cognate binding partners in tumor epithelial cells, suggesting a role in tumor-stroma crosstalk. Our findings implicate fibroblast-derived axonal-guidance genes in promoting PDAC invasion and point to a promising target for this disease. Statement of Significance Therapeutic resistance remains a major challenge in pancreatic cancer. Our findings shed light on the changes in the complex tumor microenvironment in response to chemotherapy and identify fibroblasts high in axonal-guidance genes that may drive tumor progression and chemoresistance, thus uncovering a potential avenue for targeting pancreatic cancer.

Abstract Pancreatic adenocarcinoma (PDA) has poor response to chemotherapy with a strong likelihood of cancer recurrence. While previous studies have examined patient samples collected before and after chemotherapy, few have analyzed matched samples from the same patients that allow us to deconvolute interpatient heterogeneity, a large limitation to PDA studies. Here, we study the effects of chemotherapy on tumor cells and their surrounding microenvironment using scRNA sequencing of matched pre- and post- treatment tissue biopsies from 5 patients. We utilized the Seurat v4 pipeline to observe transcriptomic differences and corresponding putative cellular interactions in both treatment states. Unsurprisingly, we found insufficient tumor epithelial cells post-treatment due to the intended cytotoxic effects of chemotherapy, leading us to focus on the tumor microenvironment where we had sufficient cells to analyze. We identified that immune cells in the TME are heterogeneously affected by chemotherapy treatment with patient specific differences, but we did identify a unanimous increase in CXCR4 in CD4 and CD8 positive T cells after treatment. Analysis of CAFs revealed that a subset of fibroblasts lose myofibroblastic features and increasingly acquire 'neural'-like features after treatment. This suggests that chemotherapy-altered fibroblasts may contribute to a pro-tumor microenvironment that facilitates recurrence through neuron–CAF–tumor interactions. Through inferred ligand-receptor pairs identified with the CellChat v2 pipeline, we observed that increased CXCL12 in these neural CAFs interacts with the increased CXCR4 on T-cells. We validated the expression of these genes in patients by staining human patient PDAC tissues (both treated and treatment naïve tissues) for neural genes that were highly enriched post-treatment in our single cell sequencing dataset. We have also begun inhibiting these genes of interest in CAFs and are exploring the effects of these ‘neural’-CAFs on patient-derived tumor organoids in culture. Citation Format: Aylin Z. Henstridge, Padma Kadiyala, Ahmed Elhoissiny, Jianhua Liu, Georgina Branch, Nandini Arya, Vaibhav Sahai, Nicole Peterson, Jorge Machicado, Richard Kwon, Allison Schulman, Erik Wamsteker, George Phillips, Martin Fernandez-Zapico, Mark Truty, Timothy Frankel, Fillip Bednar, Marina Pasca di Magliano, Costas Lyssiotis, Eileen S. Carpenter. An increase in neural cancer associated fibroblasts following exposure to chemotherapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B079.

Abstract Introduction Pancreatic adenocarcinoma (PDA) will be the second leading cause of cancer related death by 2030 and patient response to chemotherapy is poor with a strong likelihood of cancer recurrence. While past studies have investigated pre- and post-chemotherapy patient samples, there is a paucity of studies looking at fresh, patient matched samples which allow us to observe interpatient heterogeneity in chemotherapy response. Methods Here, we study the effects of chemotherapy on five different patients using sc-RNA sequencing of matched treatment naïve and post treatment tissue biopsies to compare the effects of chemotherapy, not only on the tumor cells, but also on the tumor microenvironment – a key contributor to tumor survival and proliferation. We utilized the Seurat v4 pipeline and CellChat v2 to observe transcriptomic differences in epithelial cells, myeloid cells, t-Cells, and fibroblasts and their corresponding putative interactions in pre- and post- treatment states. Results Unsurprisingly, we found insufficient tumor epithelial cells post-treatment to perform further analysis on due to the intended cytotoxic effects of chemotherapy. Therefore, we turned our focus to the tumor microenvironment, where we had sufficient numbers of stromal and immune cells, to investigate the effects of chemotherapy. We identified that fibroblasts changed the most consistently across all patients in response to chemotherapy. The fibroblasts clustered into three distinct groups: an immunomodulatory group, a myofibroblastic and neurotropic group, as well as a mixed group expressing genes from all three categories. Interestingly, the neurotropic fibroblast signature was universally enriched after chemotherapy in all five patients, despite having a small and clinically heterogeneous cohort. Conclusions Through an analysis of each fibroblast group in the treatment naïve and post treatment categories, we observed that a specific set of fibroblasts become less myofibroblastic and increasingly neurotropic following treatment, leading us to posit that fibroblasts altered by exposure to chemotherapy may potentially contribute to a ‘pro-tumor’ environment that supports recurrence. To test this in vitro, we treated patient derived fibroblasts with Gemcitabine and used RTPCR to analyze their relative changes in gene expression. We specifically probed for genes that were definitive of myofibroblasts and neurotropic fibroblasts and saw that post-chemotherapy exposure, neurotropic genes such as NEGR1 and NFIA were upregulated. This validated our scRNA conclusions that fibroblasts become increasingly neurotropic following treatment and then differentially interact with their surrounding environment. In future studies, we will further analyze the role these neurotropic fibroblasts play in chemoresponse and tumor recurrence. Matched samples are critical to studies of chemotherapy effects on PDA due to the fact that they allow us to deconvolute one aspect of PDA heterogenity - interpatient heterogeneity - which is a large limitation in studies of this disease. Citation Format: Aylin Z Henstridge, Padma Kadiyala, Ahmed M Elhossiny, Jianhua Liu, Vaibhav Sahai, Nicole Peterson, Jorge Machicado, Richard Kwon, Allison Schulman, Erik Wamsteker, George Philips, Martin Fernandez-Zapico, Mark Truty, Costas Lyssiotis, Timothy Frankel, Filip Bednar, Marina Pasca di Magliano, Eileen S Carpenter. Neurotropic fibroblast population increases following exposure to chemotherapy in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A062.