Oncolytic adeno-immunotherapy modulates the immune system enabling CAR T-cells to cure pancreatic tumors.

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients.

Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells.

Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes.IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.

The Novel Oncolytic Adenoviral Mutant Ad5-3Δ-A20T Retargeted to αvß6 Integrins Efficiently Eliminates Pancreatic Cancer Cells.

Metastatic pancreatic ductal adenocarcinomas (PDAC) are incurable due to the rapid development of resistance to all current therapeutics. Oncolytic adenoviral mutants have emerged as a promising new strategy that negates such resistance. In contrast to normal tissue, the majority of PDACs express the αvß6 integrin receptor. To exploit this feature, we modified our previously reported oncolytic adenovirus, AdΔΔ, to selectively target αvß6 integrins to facilitate systemic delivery. Structural modifications to AdΔΔ include the expression of the small but potent αvß6-binding peptide, A20FMDV2, and ablation of binding to the native coxsackie and adenovirus receptor (CAR) within the fiber knob region. The resultant mutant, Ad5-3Δ-A20T, infected and killed αvß6 integrin-expressing cells more effectively than the parental wild-type (Ad5wt) virus and AdΔΔ. Viral uptake through αvß6 integrins rather than native viral receptors (CAR, αvß3 and αvß5 integrins) promoted viral propagation and spread. Superior efficacy of Ad5-3Δ-A20T compared with Ad5wt was demonstrated in 3D organotypic cocultures, and similar potency between the two viruses was observed in Suit-2 in vivo models. Importantly, Ad5-3Δ-A20T infected pancreatic stellate cells at low levels, which may further facilitate viral spread and cancer cell elimination either as a single agent or in combination with the chemotherapy drug, gemcitabine. We demonstrate that Ad5-3Δ-A20T is highly selective for αvß6 integrin-expressing pancreatic cancer cells, and with further development, this new and exciting strategy can potentially be extended to improve the systemic delivery of adenoviruses to pancreatic cancer patients. Mol Cancer Ther; 17(2); 575-87. ©2018 AACR.

Patient-derived xenografts of gastrointestinal cancers are susceptible to rapid and delayed B-lymphoproliferation.

Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.

The hepatitis B virus X protein promotes pancreatic cancer through modulation of the PI3K/AKT signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer, with poor outcomes. Infection with the hepatitis B virus (HBV) may be associated as a worse prognosis for PDAC patients; however, the mechanisms involved in this process are unclear. We evaluated whether HBV infection leads to PDAC with a more aggressive phenotype, and attempted to elucidate the mechanisms involved. Clinicopathological data and outcomes from 64 patients with PDAC were collected and compared between serum HBsAg+ and HBsAg- patients. Furthermore, we examined the effects of the HBV X protein (HBx) on proliferation and migration of the pancreatic cancer cell lines PANC-1 and SW1990. We investigated expression changes of over 500 proteins by protein array analysis and identified several HBV- and PDAC-related candidates, which were further validated by immunoblotting and enzyme-linked immunosorbent assay. No differences in clinicopathological features were observed between HBsAg+ and HBsAg- patients; however, HBsAg+ patients had a shorter median survival time (8 vs. 13 months), although the differences were not significant. HBV DNA was detected in clinical specimens, even in PDAC patients considered "HBV-free", potentially due to occult infection. HBx expression significantly enhanced cellular proliferation and migration and induced an epithelial-mesenchymal transition phenotype. Expression of ErbB4 and TGF-α was increased in parallel with HBx expression, and several downstream pathways including PI3K/AKT, MAPK, and ERK were upregulated. Inhibition of the PI3K/AKT pathway reversed the effects of HBx in PDAC cell lines. HBx may, therefore, contribute to the progression of PDAC through modulation of these pathways.

Viro-immune therapy: A new strategy for treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease with less than 5% survival at five years. This is largely due to metastatic disease, which is already present in the majority of patients when diagnosed. Even when the primary cancer can be removed by radical surgery, local recurrence occurs within one year in 50%-80% of cases. Therefore, it is imperative to develop new approaches for the treatment of advanced cancer and the prevention of recurrence after surgery. Tumour-targeted oncolytic viruses (TOVs) have become an attractive therapeutic agent as TOVs can kill cancer cells through multiple mechanisms of action, especially via virus-induced engagement of the immune response specifically against tumour cells. To attack tumour cells effectively, tumour-specific T cells need to overcome negative regulatory signals that suppress their activation or that induce tolerance programmes such as anergy or exhaustion in the tumour microenvironment. In this regard, the recent breakthrough in immunotherapy achieved with immune checkpoint blockade agents, such as anti-cytotoxic T-lymphocyte-associate protein 4, programmed death 1 (PD-1) or PD-L1 antibodies, has demonstrated the possibility of relieving immune suppression in PDAC. Therefore, the combination of oncolytic virotherapy and immune checkpoint blockade agents may synergistically function to enhance the antitumour response, lending the opportunity to be the future for treatment of pancreatic cancer.

Oncolytic activity of avian influenza virus in human pancreatic ductal adenocarcinoma cell lines.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE: Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts.

Hepatitis B and C viruses are not risks for pancreatic adenocarcinoma.

AIM: To investigate whether hepatitis B virus (HBV) and hepatitis C virus (HCV) increase risk of pancreatic ductal adenocarcinoma (PDAC). METHODS: We recruited 585 patients with cytological and/or pathologically confirmed PDAC in National Taiwan University Hospital from September 2000 to September 2013, and 1716 age-, sex-, and race-matched controls who received a screening program in a community located in Northern Taiwan. Blood samples were tested for the presence of HCV antibodies (anti-HCV), HBV surface antigen (HBsAg), antibodies against HBsAg (anti-HBs), and hepatitis B core antigen (anti-HBc) in all cases and controls. The odds ratio (OR) of PDAC was estimated by logistic regression analysis with adjustment diabetes mellitus (DM) and smoking. RESULTS: HBsAg was positive in 73 cases (12.5%) and 213 controls (12.4%). Anti-HCV was positive in 22 cases (3.8%) and 45 controls (2.6%). Anti-HBs was positive in 338 cases (57.8%) and 1047 controls (61.0%). The estimated ORs of PDAC in multivariate analysis were as follows: DM, 2.08 (95%CI: 1.56-2.76, P < 0.001), smoking, 1.36 (95%CI: 1.02-1.80, P = 0.035), HBsAg(+)/anti-HBc(+)/anti-HBs(-), 0.89 (95%CI: 0.89-1.68, P = 0.219), HBsAg(-)/anti-HBc(+)/anti-HBs(+), 1.03 (95%CI: 0.84-1.25, P = 0.802). CONCLUSION: HBV and HCV infection are not associated with risk of PDCA after adjustment for age, sex, DM and smoking, which were independent risk factors of PDAC.

Vascular endothelial growth factor A promotes vaccinia virus entry into host cells via activation of the Akt pathway.

Vaccinia virus (VV) is an enveloped DNA virus from the poxvirus family and has played a crucial role in the eradication of smallpox. It continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer. However, the mechanisms of poxvirus entry, the host factors that affect viral virulence, and the reasons for its natural tropism for tumor cells are incompletely understood. By studying the effect of hypoxia on VV infection, we found that vascular endothelial growth factor A (VEGF-A) augments oncolytic VV cytotoxicity. VEGF derived from tumor cells acts to increase VV internalization, resulting in increased replication and cytotoxicity in an AKT-dependent manner in both tumor cells and normal respiratory epithelial cells. Overexpression of VEGF also enhances VV infection within tumor tissue in vivo after systemic delivery. These results highlight the importance of VEGF expression in VV infection and have potential implications for the design of new strategies to prevent poxvirus infection and the development of future generations of oncolytic VV in combination with conventional or biological therapies.

Generation of a novel, cyclooxygenase-2-targeted, interferon-expressing, conditionally replicative adenovirus for pancreatic cancer therapy.

BACKGROUND: Oncolytic adenoviruses provide a promising alternative for cancer treatment. Recently, adjuvant interferon (IFN)-alfa has shown significant survival benefits for pancreatic cancer, yet was impeded by systemic toxicity. To circumvent these problems adenovirus with high-level targeted IFN-alfa expression can be generated. METHODS: Conditionally replicative adenoviruses (CRAds) with improved virulence and selectivity for pancreatic cancer were generated. The vectors were tested in vitro, in vivo, and in human pancreatic cancer and normal tissue specimens. RESULTS: Adenoviral death protein and fiber modifications significantly improved oncolysis. CRAds selectively replicated in vitro, in vivo and showed persistent spread in cancer xenografts. They showed high-level replication in human pancreatic cancer specimens, but not in normal tissues. Improved IFN-CRAd oncolytic efficiency was shown. CONCLUSIONS: Optimized cyclooxygenase-2 CRAds show highly favorable effects in vitro and in vivo. We report a pancreatic cancer-specific, highly virulent, IFN-expressing CRAd, and we believe that adenovirus-based IFN therapy offers a new treatment opportunity for pancreatic cancer patients.

Hepatitis B virus status and risk of pancreatic ductal adenocarcinoma: a case-control study from China.

OBJECTIVES: This study was to assess the role of hepatitis B virus (HBV) infection in pancreatic ductal adenocarcinoma (PDAC) risk using a hospital-based case-control design. METHODS: Patients with pathologically confirmed PDAC (943) and 1128 matched controls were recruited from 2 hospitals. We evaluated the associations between risk of PDAC and age, sex, history of diabetes mellitus (DM), etc. In addition, we examined the interactive effects of HBV status and known risk factors for pancreatic cancer. RESULTS: Chronic hepatitis B and inactive hepatitis B surface antigen (HBsAg) carrier state (HBsAg positive) had a significantly increased risk of pancreatic cancer, with an adjusted odds ratio of 1.60 (95% confidence interval [CI], 1.15-2.24). Furthermore, significant interactions were detected between a history of DM and chronic hepatitis B and inactive HBsAg positive, but not with antibodies to hepatitis B core antigen (anti-HBc) positive/antibodies to HBsAg (anti-HBs) negative, with an adjusted odds ratio of 5.42 (95% CI, 2.76-10.64), compared with those who were HBsAg negative/anti-HBc negative without a history of DM. CONCLUSIONS: These results suggest that HBsAg-positive or anti-HBc-positive/anti-HBs-negative patients have an increased risk for PDAC independent of other risk factors. Significant interactions were found between a history of DM and chronic HBV infection for PDAC risk.

Enhancement of NK cell antitumor responses using an oncolytic parvovirus.

Natural killer (NK) cells play a vital role in the rejection of tumors. Pancreatic ductal adenocarcinoma (PDAC), however, remains a poor prognosis malignancy, due to its resistance to radio- and chemotherapy, and low immunogenicity. We demonstrate here that IL-2-activated human NK cells are able to kill PDAC cells. Currently, novel strategies are being pursued to combat PDAC. In this regard, oncolytic viruses, in addition to killing tumor cells, may also have the potential to augment antitumor immune responses. We found that, besides having an intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of PDAC cells. Our results show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN-γ, TNF-α and MIP-1α/ß. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors-NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected PDAC cells to NK cell-dependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. Our data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a combinatorial therapeutic approach against PDAC.

SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV.

Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.

New pancreatic carcinoma model for studying oncolytic adenoviruses in the permissive Syrian hamster.

Syrian hamster is a practical animal model for studying the systemic effects of oncolytic vectors derived from adenovirus serotype 5 (Ad5). Ad5 replicates well in Syrian hamster tissues, and Syrian hamster cell lines are available that are known to support Ad5 replication. In this study, we established four new Syrian hamster cell lines from transplantable pancreatic, renal, hepatic and lung tumors. The pancreatic cell line (SHPC6) and the renal cell line were highly permissive for Ad5 replication. The SHPC6 cell line formed disseminated intraperitoneal tumors when cells were injected into the peritoneal cavity. INGN 007, an oncolytic Ad5-based vector, completely reversed the growth of disseminated intraperitoneal SHPC6 tumor nodules following intraperitoneal injection of the vector, leading to 100% survival of the treated animals. SHPC6 cells also formed subcutaneous tumors, whose growth was suppressed by INGN 007 following intratumoral injection. INGN 007 replicated in both the intraperitoneal and subcutaneous SHPC6 tumors. Following intraperitoneal injection, INGN 007 did not replicate in the livers of hamsters with intraperitoneal SHPC6 tumors, and was not hepatotoxic. These studies suggest that the SHPC6 cell line may be useful as a model for disseminated pancreatic cancer, and that INGN 007 may be a safe and effective vector to treat these tumors.

Carcinoembryonic antigen-specific but not antiviral CD4+ T cell immunity is impaired in pancreatic carcinoma patients.

Pancreatic carcinoma is a very aggressive disease with dismal prognosis. Although evidences for tumor-specific T cell immunity exist, factors related to tumor microenvironment and the presence of immunosuppressive cytokines in patients' sera have been related to its aggressive behavior. Carcinoembryonic Ag (CEA) is overexpressed in 80-90% of pancreatic carcinomas and contains epitopes recognized by CD4(+) T cells. The aim of this study was to evaluate the extent of cancer-immune surveillance and immune suppression in pancreatic carcinoma patients by comparing the anti-CEA and antiviral CD4(+) T cell immunity. CD4(+) T cells from 23 normal donors and 44 patients undergoing surgical resection were tested for recognition of peptides corresponding to CEA and viral naturally processed promiscuous epitopes by proliferation and cytokine release assays. Anti-CEA CD4(+) T cell immunity was present in a significantly higher number of normal donors than pancreatic cancer patients. Importantly, whereas CD4(+) T cells from normal donors produced mainly GM-CSF and IFN-gamma, CD4(+) T cells from the patients produced mainly IL-5, demonstrating a skew toward a Th2 type. On the contrary, the extent of antiviral CD4(+) T cell immunity was comparable between the two groups and showed a Th1 type. The immunohistochemical analysis of tumor-infiltrating lymphocytes showed a significantly higher number of GATA-3(+) compared with T-bet(+) lymphoid cells, supporting a Th2 skew also at the tumor site. Collectively, these results demonstrate that Th2-immune deviation in pancreatic cancer is not generalized but tumor related and suggests that the skew might be possibly due to factor(s) present at the tumor site.

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.