Gutierrez-Ruiz, Omar L.

Despite the high initial responses to chimeric antigen receptor T (CART) cell therapy, most patients relapse within the first 1-2 years. T cell quality influences the success of CART cell therapies greatly, and T cells from cancer patients are prone to transition into dysfunctional states, such as exhaustion or senescence. The role of exhaustion in CART cell failures has been studied extensively; however, the role of T cell senescence has remained mostly unexplored. In this study, we aimed to determine the development of CART cell senescence and its impact on CART therapeutic efficacy. First, we studied the development of CART cell senescence using an in vitro model to induce CART cell dysfunction. Here, we cyclically activated CD19-directed chimeric antigen receptor T (CART19) cells (either costimulated via 4-1BB (BBζ) or CD28 (28ζ)). Activation cycles are designed to mimic recurrent activation of CART cells to study the impact of T cell activation on CART cell fates throughout treatment. We added a rest time in between activation cycles to focus on senesced CART cell populations since, unlike exhaustion, senescence is irreversible even after rest. We examined CART immunophenotype, cell cycle regulators, transcriptomic profile, senescence marker gene expressions and T cell effector functions on days 0 (D0-T cell), 8 (D8-baseline CART), and 15 (D15-CART cell after one activation/rest cycles). Following an activation/rest cycle, T cell exhaustion markers CTLA4, LAG3, and PD1 returned to basal levels after an initial increase due to activation, while only TIM-3 levels remained elevated (BBζ: p=0.0090, 28ζ: p=0.0394). Increased TIM-3 expression is associated with T cell senescence, which prompted us to further investigate the development of a senescence phenotype. We found a prominent T cell senescence phenotype in BBζ compared to 28ζ, as evident by increased expression of p53 (BBζ: p=0.0248), p21 (BBζ: p=0.0483), CD27 (BBζ: p=0.0080), CD28 (BBζ: p=0.0327), uPAR (BBζ vs. 28ζ: p<0.0001), and KLRG1 (BBζ vs. 28ζ: p=0.0029) in recurrently activated-rested CART cells. Next, we evaluated the transcriptomic signature of recurrently activated-rested CART cells. Gene set enrichment analysis (GSEA) indicated enrichment of gene sets related to senescent T cells as well as senMayo, a novel gene set which is reported to be selectively enriched in senescing cells or tissues, in BBζ, but not in 28ζ, compared to T cell D0 or in BBζ D15 compared to 28ζ D15 (p=0.00014 and p=0.089, respectively). MYC-related gene sets were found to be significantly enriched in recurrently activated/rested BBζ and depleted in 28ζ (p<0.1). MYC expression is increased in BBζ (p= 0.0010) while decreased in 28ζ (p=0.0036) upon recurrent activation and is higher in BBζ (D8: p=0.0018, D15: p= 0.0022) compared to 28ζ at indicated time points by qPCR. Given these results, we induced senescence in CART cells using two alternative strategies: MYC-T58A activation (oncogene-induced senescence) and irradiation. Senescence induced by MYC-T58A overexpression ameliorated only BBζ killing (p=0.0057), whereas senescence induced by irradiation improved 28ζ killing (p= 0.0086), indicating versatile impact of senescence on treatment outcome diverse CART cell therapies. Finally, analysis with two independent patient data sets correlated the emergence of senescence with contradicting clinical outcomes to BBζ and 28ζ treatments. The presence of senescent 28ζ cells in the peripheral blood of 28ζ-treated patients was reported to be associated with complete response in the clinic in an independent analysis. In another cohort, 28ζ-treated patients with higher senescence score were correlated with longer disease-free survival (p=0.0029), while senMayo gene set enrichment in BBζ-treated patients was associated with non-response in the clinic (p=0.014). In the same cohort, MYC-related gene sets-the same genes sets enriched in our recurrently activated/rested BBζ-are also enriched in nonresponsive BBζ-treated patients. In summary, our study indicates that (i) CART cells are susceptible to a state of senescence, as demonstrated using three independent approaches (repeated activation/rest, irradiation, and MYC modulation), (ii) CART senescence is more prominent in BBζ than 28ζ, and (iii) senescence is associated with worse outcomes following BBζ but not 28ζ treatment.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, CAR T-cell therapy still has low durable response rates and common associated toxicities such as cytokine release syndrome (CRS). We recently reported that sequential therapy with the CD19 monoclonal antibody tafasitamab and CD19-directed CAR T (CART19, 41BBζ costimulated) cells significantly ameliorated CAR T-cell apoptosis and tumor cell pyroptosis, improving CAR T safety and efficacy (PMID: 37879074). Building on these findings, we hypothesized that controlling massive initial CAR T-cell stimulation by diminishing extensive tumor cell pyroptosis is critical to improve CAR T safety and durability. To test this hypothesis, we evaluated several caspase (Casp) inhibitors, including Casp1, Casp3, Casp7, and Casp11, in combination with CAR T cells. We found that the Casp1 inhibitor VX765 did not inhibit CAR T antigen-specific cytotoxicity and thus focused on VX765 in subsequent assays. We first assessed CAR T proliferation upon stimulation with the CD19+ JeKo-1 mantle cell lymphoma cell line in the presence of VX765. CAR T with vehicle control showed greater proliferation at day 3, but CAR T with VX765 showed higher proliferation at day 5 (p=0.03, absolute CD3+ counts by flow cytometry). We then co-cultured CAR T and JeKo-1 for 24 hours and observed a significant reduction in apoptotic CAR T (Annexin V+ and 7-AAD- via flow cytometry) in VX765-treated conditions (p=0.05). Additionally, there was significantly reduced pyroptosis (Annexin V+ 7-AAD+) (p=0.02) but higher apoptosis (Annexin V+ 7-AAD-) in JeKo-1 in VX765-treated conditions (p=0.04). VX765-treated CAR T showed higher Bcl2 expression via flow cytometry (p=0.03). We also studied tumor cell pyroptosis by measuring the level of HMGB-1. CART19, JeKo-1, and VX765 or vehicle control were co-cultured for 24 hours, and the level of HMGB-1 was found to be significantly reduced in VX765-treated conditions, indicating reduced tumor pyroptosis (p=0.006). We then assessed the impact of Casp1 inhibition in our in vitro model of CAR T exhaustion. We stimulated CAR T with CD19+ JeKo-1 every other day for 2 weeks and then assessed CCR7 and CD45RA expression on CAR T. VX765-treated CAR T showed higher central memory (p=0.08) and naïve (p=0.02) and lower terminally differentiated effector memory phenotypes (p=0.03). We next studied the effect of Casp1 inhibition with VX765 in vivo. To recapitulate CAR T-cell toxicities, including CRS, we utilized an immunocompetent mouse model. First, we generated murine CART19 (mCART19, CD28ζ costimulated) cells. Recipient mice were then engrafted with luciferase+ murine CD19+ TBL12 cells, a murine lymphoma cell line. Mice were treated 12 days later with cyclophosphamide (300 mg/kg, ip) and 24 hours after mice were imaged and randomized to receive 1 x 107, 1) untransduced T cell control (UTD), 2) mCART19 + vehicle control, or 3) mCART19 + VX765 (25 mg/kg). VX765 was given immediately before the CART19 injection and on day 2 post-CAR T injection. Mice were serially monitored and weighed to assess CAR T-cell toxicity. Tumor burden was assessed via weekly bioluminescent imaging. Mice treated with vehicle control showed a significant reduction in weight compared to the UTD control (p=0.02), while mice treated with VX765 maintained their weight and remained active without signs of CRS, including motor weakness and hunched body. UTD mice reached humane endpoints within 3 weeks after T-cell injection. Three satellite mice from each group were euthanized and spleens were harvested. Flow cytometric analysis showed a significant reduction in apoptotic CAR T (p=) and pyroptotic tumor cells in mice treated with VX765 (p=0.03). Western blot showed lower cleaved GSDME and GSDMD in mice treated with VX765. VX765-treated mice demonstrated prolonged CAR T persistence with durable anti-tumor activity. In summary, Casp1 inhibition with VX765 significantly reduced CAR T apoptosis, improved memory phenotype, and increased delayed CAR T proliferation. VX765 also reduced tumor cell pyroptosis and CRS incidence without impacting CAR T anti-tumor activity in vivo. Given the favorable preclinical outcomes and existing clinical data on VX765, this approach holds substantial translational potential for enhancing the safety and efficacy of CAR T-cell therapy in patients with large B-cell lymphoma.

Abstract While chimeric antigen receptor T (CART) cell therapy has shown remarkable success, the development of exhaustion limits durable response. We identified a role for interleukin (IL)-4 in the development of CART cell exhaustion through three independent approaches including: 1) a genome-wide CRISPR knockout screen using healthy donor CART cells in an in vitro model for exhaustion, 2) RNA and ATAC sequencing on freshly produced and chronically stimulated healthy donor CART cells, and 3) RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel. Further, in vitro validation studies revealed that CD19 directed CART (CART19) cells chronically stimulated in the presence of human recombinant IL-4 (hrIL-4) displayed signs of exhaustion such as 1) decreased proliferation (p = 0.01), 2) increased coexpression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and interferon (IFN)-γ (p= 0.02, p = 0.002). Encouragingly, CART19 cells combined with an IL-4 monoclonal antibody improved antitumor activity (p = 0.045) and expansion (p = 0.01) while also decreasing the co-expression of inhibitory receptors (p = 0.02) in a mantle cell lymphoma xenograft mouse model. Building on these results, we asked if IL-4 driven exhaustion results from a direct impact of IL-4 on CART cells. To test this, we used a tumor-free assay where CART19 cells were chronically stimulated with CD19 beads in the presence of hrIL-4 or diluent. CART cells treated with hrIL-4 displayed an exhausted phenotype characterized by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 (p = 0.01). Next, we asked if IL-4 driven CART cell exhaustion is dependent on the costimulatory domain. We tested the impact of IL-4 on both CD28ζ and 41BBζ costimulated CART19 cells. Similar to our previous studies with CART19-28ζ cells, chronic stimulation of CART19-BBζ cells in the presence of hrIL-4 enhanced the exhausted phenotype as seen by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 and IFN-γ (p = 0.08 and p = 0.007). Finally, we asked if IL-4 induces exhaustion independently of its classic role in Th2 polarization of CD4 CART cells. Following CART production, we isolated CD8 cells and chronically stimulated them in the presence of hrIL-4 or diluent. CD8 CART cells treated with hrIL-4 displayed an enhanced exhausted profile as seen by 1) decreased proliferative ability (p < 0.0001), 2) increased co-expression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and IFN-γ (p < 0.0001, p = 0.004). Together, our data indicates a novel role for IL-4 in the development of CART cell exhaustion that is independent of tumor cells, costimulatory domain, and CD4 cells. As such, we believe IL-4 neutralization may be a widely applicable and actionable approach to improve the durable response to CART cell therapy. Citation Format: Carli M. Stewart, Elizabeth L. Siegler, Truc N. Huynh, R. Leo Sakemura, Brooke Kimball, Long Mai, Kun Yun, James H. Girsch, Jennifer Feigin, Omar Gutierrez Ruiz, Makena Rodriguez, Ekene Ogbodo, Ismail Can, Claudia Manriquez Roman, Olivia Sirpilla, Hong Xia, Jenny Kim, Justin Budka, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 drives CART cell exhaustion in a CD4 independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 37.

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3841.

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3849.

Our group has reported that transient CD19 antigen masking of leukemic cells with the CD19 monoclonal antibody (mAb), tafasitamab, resulted in reduction of CD19 chimeric antigen receptor T cell (CART19) apoptosis, enhanced CAR T proliferation, improved anti-tumor activity, diminished tumor pyroptosis, and reduced severity of cytokine release syndrome (CRS) in preclinical models. In this report, we aimed to study the mAb characteristics responsible for these observations. Specifically, we hypothesized that binding affinity of CD19 mAbs might affect the subsequent activation and apoptosis of CART19 as well as tumor cell pyroptosis in preclinical models. To test our hypothesis, we first determined the affinities of the CD19-targeting antibodies tafasitamab, HD37 and FMC63 to recombinant human CD19 protein. Tafasitamab demonstrated the strongest binding affinity among the three tested antibodies [tafasitamab ( K D: 1.0±0.2 nM), HD37 ( K D: 4.1±0.3 nM), FMC63 ( K D: 3.4±1.2 nM)]. Next, we studied CART19 apoptosis in the presence of these CD19 mAbs with different binding affinities. Here, we co-cultured CART19, CD19 + mantle cell lymphoma JeKo, and 400 μg/mL of CD19 mAb for 1 hour and measured CD3 + 7-AAD - Annexin V + T cells with flow cytometry. The number of apoptotic CART19 cells was significantly reduced by tafasitamab co-treatment compared to HD37, FMC63, or control IgG [control IgG (mean = 19.1, standard deviation (SD) = 0.8), tafasitamab (mean = 13.1, SD = 2.9), HD37 (mean = 17.8, SD = 0.8), FMC63 (mean = 18.4, SD = 1.0)]. We also performed an in vitro high mobility group box 1 (HMGB1) assay to assess if different affinities of the CD19 mAbs affect tumor cell pyroptosis triggered by CART19. Here, we co-cultured CART19, CD19 + JeKo, and 400 μg/mL of CD19 mAb for 24 hours and assessed HMGB1 on JeKo. In analogy to the apoptosis data, tafasitamab was the only mAb that prevented tumor cell pyroptosis, indicated by reduced HMGB1 expression (Fig 1A). Given these results, we aimed to study the impact of CD19 occupancy with different CD19 mAbs prior to CART19 infusion on tumor pyroptosis in vivo. We utilized a mouse model that mimics CAR T toxicities. Immunocompromised NSG mice were inoculated via tail vein with 5x10 6 leukemic cells derived from patients with relapsed/refractory acute lymphoblastic leukemia. Two weeks later (day -7), tumor burden was assessed by peripheral blood sampling. Leukemic cells were defined as murine CD45 -, human CD45 + CD19 + CD20 + via flow cytometry, and volumetric absolute number was calculated. Mice were then randomized according to tumor burden to receive IgG control [Group 1 (G1)], tafasitamab (G2), HD37 (G3), or FMC63 (G4) (5 mg/kg, ip) at day -7. CRS was determined by weight loss, development of motor weakness, poor body posture, and elevated serum cytokine levels. At day 0, all mice received 3x10 6 luciferase + CART19 via tail vein. Mice were monitored with daily bioluminescent imaging (BLI) to track and quantify CART19 expansion and physical assessment to detect toxicity. BLI on day 4 revealed that CART19 expansion in the tafasitamab-treated group (G2, mean = 1x10 7, SD = 2.3x10 7) was significantly lower than in the control group (mean = 3.5x10 7, SD = 9.3x10 7), the HD37-treated group (G3, mean = 4x10 7, SD = 6.6x10 7), and the FMC63-treated group (G4, mean = 2x10 7, SD = 3.59x10 7), [G1 vs G2: 95% confidence interval (CI) 3.5x10 7 to 1.1x10 8, p <0.0001, G1 vs G3: 95% CI -7.5x10 7 to -1.1x10 8, p=0.005, G1 vs G4: 95% CI -4.5x10 7 to 1.9x10 7, p=0.71] indicating that tafasitamab pretreatment significantly reduced early activation of CART19 compared to IgG control and HD37 in this model. Mice were bled on day 4 to analyze plasma cytokine. Tafasitamab-treated mice (G2) secreted significantly less IFNγ (Fig 1B), IL10 (Mean G1: 78, G2: 2.52, G3: 12.2, G4: 13.9 pg/mL), MDC (Mean G1: 4072, G2: 445, G3: 921, G4: 1364 pg/mL), and MIP1β (Mean G1: 283, G2: 59.1, G3: 148, G4: 123 pg/mL) compared to G1, G3, and G4. Satellite mice were euthanized on day 1 and spleens were harvested. Western blot analysis of splenocytes revealed that G1, 3, and 4 showed significantly lower expression of apoptosis-inducing protein gasdermin E on leukemic cells compared to G2, indicating reduced tumor pyroptosis in G2. In summary, application of the high affinity mAb tafasitamab prior to CART19 resulted in reduced CAR T apoptosis, diminished tumor cell pyroptosis, and reduction of CRS compared to lower affinity CD19 mAbs in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality worldwide. This is largely due to the lack of routine screening protocols, an absence of symptoms in early-stage disease leading to late detection, and a paucity of effective treatment options. Critically, the majority of patients either present with metastatic disease or rapidly develop metastatic disease. Thus, there is an urgent need to deepen our understanding of metastasis in PDAC. During metastasis, tumor cells escape from the primary tumor, enter the circulation, and travel to a distant site to form a secondary tumor. In order to accomplish this relatively rare event, tumor cells develop an enhanced ability to detach from the primary tumor, migrate into the surrounding matrix, and invade across the basement membrane. In addition, cancer cells interact with the various cell types and matrix proteins that comprise the tumor microenvironment, with some of these factors working to promote metastasis and others working to suppress it. In PDAC, many of these processes are not well understood. The purpose of this review is to highlight recent advances in the cell biology of the early steps of the metastatic cascade in pancreatic cancer. Specifically, we will examine the regulation of epithelial-to-mesenchymal transition (EMT) in PDAC and its requirement for metastasis, summarize our understanding of how PDAC cells invade and degrade the surrounding matrix, and discuss how migration and adhesion dynamics are regulated in PDAC to optimize cancer cell motility. In addition, the role of the tumor microenvironment in PDAC will also be discussed for each of these invasive processes.