From precursor to cancer: decoding the intrinsic and extrinsic pathways of pancreatic intraepithelial neoplasia progression.

Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.

Pancreatic cancer is the fourth most common cause of cancer death in the United States. It remains a highly lethal malignancy despite surgical and chemotherapeutic advances. In spite of progress made delineating the mechanisms underlying pancreatic cancer, there has been little improvement in survival rates over the past fifty years. Copy number analysis of human invasive pancreatic ductal adenocarcinoma (PDAC) revealed massively rearranged cellular karyotypes as well as significant clonal complexity. Given the genetic complexity of already invasive PDAC, we believe it will be important to understand earlier events in pancreatic cancer, i.e. PanIN formation and progression. We hypothesized that stage-specific bottlenecks may lead to clonal selection during pancreatic intraepithelial neoplasia (PanIN) formation and progression. In order to test this hypothesis, we examined evolving clonal complexity during the progression of pancreatic intraepithelial neoplasia in the Mist1Tg/wt; LSL-KrasG12D (KCiMist1) mouse model of pancreatic cancer. Following KrasG12D activation in adult acinar cells, these mice develop acinar to ductal metaplasia (ADM) and preinvasive PanIN 1 - PanIN 3 lesions in a manner that faithfully recapitulates the human disease. Our strategy involves crossing MistTg/wt; LSL-KrasG12D to Brainbow2.1Tg/Tg “Confetti” mice, in which individual cells undergo stochastic recombination of the Confetti reporter to activate expression of either nuclear GFP, membrane-associated CFP, cytoplasmic RFP or cytoplasmic YFP. Following tamoxifen induction, mice were given cerulein injections to increase PanIN density, and sacrificed at four and nine weeks post-tamoxifen. Following additional immunofluorescent labeling with EpCAM to demarcate individual cells, confocal imaging was performed. Lesions were characterized by grade (ADM, early PanIN and late PanIN), and the number of clones, defined as adjacent cells sharing an identical fluorescent protein signature, was compared at 4 and 9 weeks. For AMDs, early PanIN and late PanIN, we observe both monoclonal and polyclonal lesions. Our analysis of evolving clonal complexity over time is ongoing, but preliminary data suggests a time-dependent decrease in clonal complexity. The average number of clones seen in individual ADM and late PanIN lesions decreased in the mice sacrificed at nine week post tamoxifen induction compared to those sacrificed at four weeks post tamoxifen induction (1.5 +/- 0.52 clones per lesion vs. 2.29 +/- 1.14 clones per lesion for ADM and 2.5 +/- 1.29 vs. 3.75 +/- 1.89 clones per lesion for late PanIN). In contrast, there was little difference in clonal complexity noted for early PanINs at 4 vs. 9 weeks (2.17 +/- 1.19 clones per lesion in the four week cohort vs. 2.42 +/- 1.24 clones per lesion in the nine week time point). These preliminary data suggest an overall trend towards decreased clonal complexity of PanIN lesions over time, suggesting significant selective pressure at even the earliest stages of pancreatic cancer. This abstract is also presented as Poster A62. Citation Format: Kelly J. Lafaro, Audrey Hendley, Jennifer Bailey, Steven Leach. Clonal composition and clonal selection during PanIN progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR04.

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Finding and Killing the CRABs of Pancreatic Cancer

Despite advances in our knowledge of human pancreatic ductal adenocarcinoma (PDAC), targeted therapies have not yet significantly translated to an improved overall survival for patients. Pancreatic tumor microenvironment enriched by infiltrating immune cells and consisting of dense fibrotic stroma is characterized by desmoplasia, the major contributors of tumor-associated inflammation. Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is widely accepted as a key player in the TNF-α and TGF-β -induced signaling, and principal contributor of tumor fibrosis, inflammation and cell proliferation. Here, we show that pancreatic tumors over- express TAK1, and are associated with fibrotic-stroma and infiltrating immune cells. Also, p-TAK1(ser412) expression is correlated with progression of pancreatic intraepithelial neoplasia (PanIN) lesions to PDAC. Thus, we hypothesized TAK1 as an important target for inhibition of the tumor-associated fibrosis, inflammation and PanIN progression to PDAC. To prove above hypothesis, we tested a selective TAK1 inhibitor, Takinib for its toxicity and efficacy against PanIN progression in the inducible Ptf1CreERT.LSL-KrasG12D (KC) mouse model. Takinib was synthesized and fed to wild type C57BL/6J mice (n=6) at 250 ppm and 500ppm in diet for 6 weeks to determine toxicity. Bodyweight gain, organ weights and serum enzyme analysis did not indicate any toxicity at the tested doses. For efficacy study, LSL-KrasG12D mice were bred with Ptf1-CreERTmice. Pups were genotyped and randomized to groups (n=12). PanINs -PDAC was induced in the KC mice by tamoxifen (oral gavage) followed by 250ppm Takinib administration in diet for 20 weeks. After termination, pancreas tissue sections were evaluated for PanIN multiplicity and PDAC incidence/spread. Administration of Takinib led to significant reduction in PanIN 1 by 54% (p<0.02), PanIN 2 by 77% (p<0.001) and PanIN 3 by 80% (p<0.02) in the KC mice compared to untreated mice. PDAC incidence was also reduced with an increase in normal pancreas in Takinib administered KC mice. Taken together our results suggest that TAK1 is a valuable target for PDAC, and Takinib possesses efficacy against the PanIN and PDAC progression in the KC mouse model warranting further studies. (Supported by PHF Team Science grant and Kerley-Cade Endowed Chair). Citation Format: Venkateshwar Madka, Gopal Pathuri, Gaurav Kuma, Anil Singh, Nicole Stratton, Courtney Houchen, Altaf Mohammed, Chinthalapally V. Rao. Targeting TAK1 for intercepting PanIN progression to ductal adenocarcinoma in an inducible KC mouse model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A014.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with limited treatment efficacy. Advances in precision oncology, enabled by next-generation sequencing, have highlighted key molecular targets. Kirsten rat sarcoma viral oncogene homolog mutations, present in up to 90% of cases, drive aggressive biology, though most variants remain undruggable; allele-specific inhibitors and exosome-based RNA interference are under exploration. Breast cancer susceptibility gene 1/2 mutations occur in 4%-7% of patients, conferring sensitivity to platinum agents and poly(ADP-ribose) polymerase inhibitors. Other rare but actionable alterations - such as v-raf murine sarcoma viral oncogene homolog B1 (V600), neurotrophic tyrosine receptor kinase, fibroblast growth factor receptor 2, and RET fusions - show benefit in tumor-agnostic trials, broadening options for selected subgroups. Immunotherapy is limited, as high tumor mutational burden and mismatch repair deficiency are uncommon in PDAC, though predictive when present. Co-mutations in tumor protein p53, cyclin-dependent kinase inhibitor 2A, and SMAD4 further stratify prognosis and influence therapy response. Cross-cancer analyses underscore the necessity of PDAC-specific strategies despite shared genomic drivers. Collectively, these insights support routine germline and somatic testing, enrollment in biomarker-matched trials, and rational combination strategies, establishing molecular profiling as central to advancing precision treatment in pancreatic cancer.

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic‐clear 3D histology is used to analyze entire pancreases of 2‐week‐old Pdx1‐Cre; LSL‐KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN‐associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+‐driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

To increase the knowledge about the development of pancreatic ductal adenocarcinoma, (PDAC) detailed analysis of the tumor progression is required. To identify proteins differentially expressed in the pancreatic intraepithelial neoplasia (PanIN), the precursor lesions of PDAC, we conducted a quantitative proteome study on microdissected PanIN cells. Proteins from 1000 microdissected cells were subjected to a procedure combining fluorescence dye saturation labeling with high resolution two-dimensional gel electrophoresis (2-DE). Differentially regulated protein spots were identified using protein lysates from PDAC tissues as a reference proteome followed by nanoLC-ESI-MS/MS. Thirty-seven single lesions of different PanIN grade (PanIN 1A/B, PanIN 2, PanIN 3) from nine patients were analyzed. Their protein expression was compared with each other, with PDAC cells and with normal ductal cells. The differential expression of differentially regulated protein spots was validated by means of immunohistochemistry using tissue microarrays. Of 2500 protein spots, 86 were found to be significantly regulated (p < 0.05, ratio > 1.6) during PanIN progression. Thirty-one nonredundant proteins were identified by mass spectrometry. Immunohistochemistry revealed that the differential expression of the selected candidate proteins major vault protein (MVP), anterior gradient 2 (AGR 2) and 14-3-3 sigma, annexin A4, and S100A10 could be successfully validated in PanIN lesions. The highly sensitive and robust proteome analysis revealed differentially regulated proteins involved in pancreatic tumor progression. The analysis of normal preneoplastic and neoplastic pancreatic tissue establishes a basis for identification of candidate biomarkers in PanIN progression that can be detected in pancreatic juice and in serum or are candidates for in vivo imaging approaches.

The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic. Non‑synonymous SNVs were analyzed with SNPs&GO and PROVEAN. A total of 13 samples were analyzed by high resolution copy number analysis on an Affymetrix SNP array 6.0. RNAseq resulted in an average of 264 coding non‑synonymous novel SNVs (ranging from 146‑374) and 16 novel insertions or deletions (In/Dels) (ranging from 6‑24) for each sample, of which a mean of 11.2% were disease‑associated and somatic events, while 34.7% were frameshift somatic In/Dels. From this analysis, alterations in the known oncogenes associated with PDAC were observed, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (93.7%) and inactivation of cyclin‑dependent kinase inhibitor 2A (CDKN2A) (50%), mothers against decapentaplegic homolog 4 (SMAD4) (50%), and tumor protein 53 (TP53) (56%). One case that was negative for KRAS exhibited a G13D neuroblastoma RAS viral oncogene homolog mutation. In addition, gene fusions were detected in 10 samples for a total of 23 different intra‑ or inter‑chromosomal rearrangements, however, a recurrent fusion transcript remains to be identified. SNP arrays identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in the chromosome arms 6p, 12p, 18q and 19q which contain KRAS, GATA binding protein 6, protein kinase B and cyclin D3. Deletions were identified on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q and 19q which involve TP53, CDKN2A/B, SMAD4, runt‑related transcription factor 2, AT‑rich interactive domain‑containing protein 1A, phosphatase and tensin homolog and serine/threonine kinase 11. In conclusion, genetic alterations in PDCA were observed to involve numerous pathways including cell migration, transforming growth factor‑β signaling, apoptosis, cell proliferation and DNA damage repair. However, signaling alterations were not observed in all tumors and key mutations appeared to differ between PDAC cases.

Pancreatic ductal adenocarcinoma (PDAC) originates from precursor lesions, the most common of which are known as pancreatic intraepithelial neoplasia (PanIN). PanIN formation and progression is accompanied by accumulation of a precursor microenvironment (PME) composed of desmoplastic stroma that plays an important role in promoting tumor growth and immune evasion. The crosstalk between tumor cells and stromal cells is an area of active investigation. While PanIN and the PME have been extensively studied in mouse models, their nature in humans have remained elusive as there is no clinical indication to sample healthy pancreas. Through a unique collaboration with the Gift of Life Organ and Tissue donation program, we performed transcriptomic analysis on normal pancreata declined for transplant. We noted that epithelial cells in the PanIN and cancer microenvironment were transcriptomically similar; however, their surrounding stromal fibroblasts were markedly distinct. Given that most precursor lesions remain quiescent while some progress to tumor, we sought out to elucidate how the transcriptional programs in epithelial cells influence fibroblast cell fate. To do so, we developed a 3D ex-vivo organoid-fibroblast coculture system using primary organoid and fibroblast cultures successfully derived from Gift of Life pancreata or PDAC patients undergoing surgical resection. Our coculture system retains histological features of heterogeneity observed in vivo. Using single cell RNA-sequencing analysis of matched fibroblast-organoid cocultures we identified factors that were highly expressed in fibroblasts in tumor cocultures, but not in normal cocultures. We are currently interrogating candidate ligand/receptor interactions in our cocultures to determine the mediators of this epithelial-mediated fibroblast polarization. This model will allow us to gain an understanding about how components of the microenvironment restrain or promote malignant transformation and serve as a great tool to study therapies targeting tumor and stromal cells in a patient specific manner. Citation Format: Padma Kadiyala, Ahmed Elhossiny, Jianhua Liu, Alexander Bray, Katelyn Donahue, Arvind Rao, Jiaqi Shi, Yaqing Zhang, Filip Bednar, Timothy Frankel, Eileen Carpenter, Marina Pasca Di Magliano. Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment [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 A057.

Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic cancer still remains a devastating disease almost uniformly lethal with a &amp;lt;6% five year survival, despite tremendous scientific efforts for over three decades. Developing novel strategies to prevent or delay progression of pancreatic cancer is currently of intense interest. Clinical and preclinical studies have clearly demonstrated eflornithine (DFMO) as a potential chemopreventive agent for several cancers. However, efficacy of DFMO in a preclinical model that recapitulates human pancreatic cancer has not yet been evaluated. The rationale for the use of DFMO as a cancer chemopreventive agent has been strengthened in recent years because ODC has been shown to be transactivated by the c-myc oncogene in cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. In the present study, we tested the effects of eflornithine on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC) in Kras activated p48Cre/+-LSL-KrasG12D/+ transgenic mice. Six-week old male and female KrasG12D/+ (∼35/group) mice were fed (AIN-76A) diets containing 0, 1000 and 2000 ppm DFMO for 38 weeks. No significant body weight differences were observed within the treatment groups. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, apoptosis and cell cycle makers; PCNA, p21, cyclin E, Bcl-xl, c-myc, caveolin-1, cyclin D1 and β-catenin by IHC, Western blotting, and/or RT-PCR methods. Results suggests that control diet fed mice showed 85 and 65% incidence of PDAC in male and female mice, respectively. Whereas 1000 ppm DFMO diet fed mice showed 10% (male) and 16% (female) incidence of PDAC respectively; and, 2000 ppm DFMO diet fed mice showed 6% (male) and 9% (female) incidence of PDAC respectively. The pancreatic tumor weights were decreased by 31-43% (p&amp;lt;0.03-0.001) with both doses of DFMO in male and female mice. Most importantly, the drug treatment showed &amp;gt;95% (p&amp;lt;0.07-0.0006) inhibition of carcinoma spread in the pancreas. Also, a significant dose dependent suppression of PanIN 3 lesions (carcinoma in-situ) (27 and 31% P&amp;lt;0.04 in male; 21 and 33% in female) was observed in mice fed with DFMO at 1000 and 2000 ppm, respectively. The pancreas of mice fed DFMO diets showed a significant inhibition of PCNA, cyclin D1, β-catenin, cyclin E, Bcl-xl, c-myc, cavolin-1 expression levels (p&amp;lt;0.05-0.0002); and increased p21 and apoptosis, when compared to the pancreatic cancer tissues derived from control diet fed mice. In summary, our preclinical data clearly indicate that DFMO has significant potential for undertaking clinical trials of pancreatic cancer chemoprevention. {Supported by NCI-CN-N01-53300}. Citation Format: Altaf Mohammed, Naveena B. Janakiram, Misty Brewer, Rebekah L. Ritchie, Anuj Marya, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Eflornithine (DFMO) prevents progression of pancreatic intraepithelial neoplasia to ductal adenocarcinoma in LSL-KrasG12D/+ mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B115.

Pancreatic Ductal Adenocarcinoma (PDA) is the most common form of pancreatic cancer driven primarily by mutations in V‐Ki‐Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (Kras) gene. Its most common mutation, G12D, cause KRAS to remain constitutively active. KrasG12D expressing pancreatic acinar cells undergo transdifferentiation into ductal phenotype and form early tumorigenic lesions called Pancreatic Intraepithelial Neoplasm (PanIN). Coincidentally occurring inactivating mutations of tumor suppressor genes, such as Cyclin‐dependent Kinase Inhibitor 2A (Cdkn2a), Tumor Protein p53 (Tp53), and Mothers Against Decapentaplegic Homolog 4 (Smad4) trigger progression of PanINs through stages from 1A to 3, eventually leading to carcinoma in situ and metastasis.G‐protein Coupled Receptors (GPCR) are transmembrane receptors that transfer extracellular signals to intracellular heterotrimeric G‐proteins with Gα and Gβγ subunits. Upon activation, dissociated Gα and Gβγ subunits separately induce intracellular second messengers for key cellular responses involved in various aspects of development, metabolism, and cell motility. GPCR signaling is negatively regulated by intracellular Regulator of G‐protein Signaling (RGS) proteins that deactivate G‐protein by mediating re‐association of Gα and Gβγ subunits.Although GPCRs are the largest non‐antibiotic drug targets, their specific role in pancreatic cancer is not much known. GPCR signaling can influence RTK pathways in cross‐talk via various second messengers. The interplay between these two major classes of cellular pathways employing both heterotrimeric and monomeric G‐proteins has therefore potential to shed new light on pancreatic cancer research.We have previously identified Rgs16 expression as a marker of pancreatic tumor formation in Rgs16::GFP o/o; Ptf1aCre/+; Lox‐STOP‐Lox‐KrasG12D/+; Cdkn2af/f (Rgs16::GFP‐KIC) mice from the onset of earliest PanINs at two weeks of age (Dis. Model Mech. 8, 1201). Using ImageJ software to quantitatively determine eGFP expression in micrographs, we have developed a rapid in vivo assay (RIVA) where we represent pancreatic tumor burden by one month of age and compare outcomes of known and novel therapeutics following two week long drug regimens. We found that just like the recently developed drug, BGB324, targeting an important RTK, called Anexelekto (Axl), the anticoagulant warfarin was more effective in reducing tumor burden than standard chemotherapy alone.Currently, my research focus is centered on understanding active GPCR pathways that stimulate cell growth, survival, and migration by enhancement of Kras downstream signaling. My aim is comparison of the effects of general G‐protein signaling inhibition in Kras mutant and wild‐type cell lines, such as AsPC‐1 in contrast to BxPC‐3, on cell behavior and making connections to active GPCR signaling components.Support or Funding InformationSupported by NCI CA161624.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OBJECTIVES A proportion of resectable pancreatic ductal adenocarcinoma (PDAC) patients display poorer survival due to profound local immune suppression. However, a pathological/morphological parameter that could functionally read out immune evasion and predict patient survival has not been defined. This study investigated the feasibility of heterotypic cell-in-cell (CIC) structures for immune cell cannibalism by tumor cells to serve as a parameter for survival prediction in resectable PDAC patients. METHODS A total of 410 samples from PDAC patients were examined using the methods of “EML” multiplex staining or immunohistochemistry (IHC). Prognostic CIC candidates were initially identified in samples plotted in tissue microarray (n=300), then independently validated in specimens from the First Affiliated Hospital of Sun Yat-Sen University (n=110). The Kaplan–Meier estimator and/or the Cox regression model were used for univariate and multivariate analysis. A nomogram was made using the Regression Modeling Strategies. RESULTS CICs were prevalent in cancerous (203/235) but not non-malignant tissues (15/147). Among the 4 CIC subtypes identified, 2 heterotypic subtypes with tumor cells internalizing CD45+ lymphocytes (LiT, mOS = 8 vs. 14.5 months, p = 0.008) or CD68+ monocytes (MiT, mOS = 7.5 vs. 15 months, p = 0.001), and overall CICs (oCIC, mOS = 10 vs. 27 months, p = 0.021), but not homotypic CICs (TiT, p = 0.089), were identified in univariate analysis as adverse prognostic factors of overall survival (OS) of PDAC. Notably, through cannibalism of immune cells by tumor cells, heterotypic CICs (L/MiT: LiT plus MiT) could independently predict shorter OS (HR = 1.85, p = 0.008) in multivariate analysis, with a performance comparable or even superior to traditional clinicopathological parameters such as histological grade (HR = 1.78, p = 0.012) and TNM stage (HR=1.64, p = 0.108). This was confirmed in the validation cohort, where L/MiT (HR = 1.71, p = 0.02) and tumor–node–metastasis (TNM) stage (HR = 1.66, p = 0.04) were shown to be independent adverse prognostic factors. Moreover, L/MiT stood out as the most prominent contributor in nomogram models constructed for survival prediction (area under the curve = 0.696 at 14 months), the dropout of which compromised prediction performance (area under the curve = 0.661 at 14 months). Furthermore, stratification analysis indicated that L/MiT tended preferentially to impact young and female patients (HR = 11.61, p CONCLUSION This was the first CIC profiling to be performed in PDAC, and is currently largest for human tumors. Subtyped CICs, as a valuable input to the traditional variables such as TNM stage, represent a novel type of prognostic factor. The formation of heterotypic L/MiT may be a surrogate for local immune evasion and predict poor survival, particularly in young female patients of resectable PDAC. Study Highlights Prior knowledge The post-operation survival periods of resectable pancreatic ductal adenocarcinoma (PDAC) patients range widely, and the search for reliable prognostic biomarkers is warranted. Although profound local immune suppression is implicated in PDAC progression and poor patient survival, a prognostic marker to read immune evasion in situ is not yet available. The impact of subtyped cell-in-cell (CIC) structures, which target either tumor or immune cells for internalization and death, on PDAC patient survival is not clear. Novelty of study This study presents the first CIC subtype profiling in PDAC, which is currently the largest of its type for human cancers. Subtyped CIC structures were identified and confirmed independently as a valuable prognostic factor for PDAC patients, with a performance comparable or superior to traditional variables such as tumor–node–metastasis (TNM) stage. The L/MiT heterotypic CIC subtype, surrogating a type of cellular immune evasion, could independently predict poor survival, particularly for young female patients of resectable PDAC.