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Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a dismal survival rate. Screening the general population for early detection of PDAC is not recommended, but because early detection improves survival, high-risk individuals, defined as those meeting criteria based on a family history of PDAC and/or the presence of known pathogenic germline variant genes with PDAC risk, are recommended to undergo screening with MRI and/or endoscopic ultrasound at regular intervals. The Pancreatic Cancer Early Detection (PRECEDE) Consortium was formed in 2018 and is composed of gastroenterologists, geneticists, pancreatic surgeons, radiologists, statisticians, and researchers from 40 sites in North America, Europe, and Asia. The overarching goal of the PRECEDE Consortium is to facilitate earlier diagnosis of PDAC for high-risk individuals to increase survival of the disease. A standardized MRI protocol and reporting template are needed to enhance the quality of screening examinations, improve consistency of clinical management, and facilitate multiinstitutional research. We present a consensus statement to standardize MRI screening and reporting for individuals with elevated risk of pancreatic cancer.

Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular environment. Studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition, metastasis, angiogenesis, and mediating the interaction of tumor cells and microenvironment. A systematic characterization of EVs from pancreatic cancer cells and cancer-associated fibroblasts (CAFs) would be valuable for studying the roles of EV proteins in pancreatic tumorigenesis.Proteomic and functional analyses were applied to characterize the proteomes of EVs released from 5 pancreatic cancer lines, 2 CAF cell lines, and a normal pancreatic epithelial cell line (HPDE).More than 1400 nonredundant proteins were identified in each EV derived from the cell lines. The majority of the proteins identified in the EVs from the cancer cells, CAFs, and HPDE were detected in all 3 groups, highly enriched in the biological processes of vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, including epithelial-mesenchymal transition, complement, and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs.These findings support the roles of EVs as a potential mediator in transmitting epithelial-mesenchymal transition signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development.

Genetic testing is recommended for all pancreatic ductal adenocarcinoma (PDAC) patients. Prior research demonstrates that multidisciplinary pancreatic cancer clinics (MDPCs) improve treatment- and survival-related outcomes for PDAC patients. However, limited information exists regarding the utility of integrated genetics in the MDPC setting. We hypothesized that incorporating genetics in an MDPC serving both PDAC patients and high-risk individuals (HRI) could: (1) improve compliance with guideline-based genetic testing for PDAC patients, and (2) optimize HRI identification and PDAC surveillance participation to improve early detection and survival.Demographics, genetic testing results, and pedigrees were reviewed for PDAC patients and HRI at one institution over 45 months. Genetic testing analyzed 16 PDAC-associated genes at minimum.Overall, 969 MDPC subjects were evaluated during the study period; another 56 PDAC patients were seen outside the MDPC. Among 425 MDPC PDAC patients, 333 (78.4%) completed genetic testing; 29 (8.7%) carried a PDAC-related pathogenic germline variant (PGV). Additionally, 32 (9.6%) met familial pancreatic cancer (FPC) criteria. These PDAC patients had 191 relatives eligible for surveillance or genetic testing. Only 2/56 (3.6%) non-MDPC PDAC patients completed genetic testing (p 0.01). Among 544 HRI, 253 (46.5%) had a known PGV or a designation of FPC, and were eligible for surveillance at baseline; of the remainder, 15/291 (5.2%) were eligible following genetic testing and PGV identification.Integrating genetics into the multidisciplinary setting significantly improved genetic testing compliance by reducing logistical barriers for PDAC patients, and clarified cancer risks for their relatives while conserving clinical resources. Overall, we identified 206 individuals newly eligible for surveillance or genetic testing (191 relatives of MDPC PDAC patients, and 15 HRI from this cohort), enabling continuity of care for PDAC patients and at-risk relatives in one clinic.

CD44 regulation of Snail expression.

Effects of trametinib and palbociclib alone and in combination on growth and expression of various signaling proteins

XLSX file - 776K, Breast sample kinome compendium by RNA-Seq.

XLSX file - 613K, RNA-Seq data for matched primary pancreatic adenocarcinoma xenograft tissue, DS-08-947, and derived xenograft cell line.

Supplementary Materials and Methods; Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse hedgehog proteins using surface plasmon resonance. Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and osteoblast differentiation in a dose-dependent manner. Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2 cells by hedgehog antibodies. Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft stromal cells but not tumor cells. Supplementary Figure S5. Hedgehog pathway component RNA expression in primary pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived from pancreatic explant model 947. Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats. Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats. Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology study in cynomolgus monkeys. Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.

CD44 regulates pancreatic cancer invasion and metastasis.

Supplementary Table 1. Percentage of CA-MSCs (CD44+CD73+CD49α+ CD90+) from primary human pancreatic cancer derived CAF cultures and accompanying clinical data. Supplementary Table 2. Extent of local invasion, lung tumor emboli, and liver metastasis in tumors that were derived from injections of tumor cells alone, tumor cells + CAF, and tumor cells + CA-MSCs (n=8 per group) at six weeks post implantation. Supplementary Table 3. Percentage of CD10 expressing CA-MSCs from primary human pancreatic cancer derived CAF cultures.

Supplementary table and figure legends

Legends for figures S1-S3

COX-2 expression at the RNA and protein levels

Supplementary Figure 2. Pancreatic cancer associated MSCs promote tumor cell growth and invasion. A. GFP-labeled tumor cells were grown alone or co-cultured with CA-MSCs or CAFs. GFP-labeled tumor cells were grown in growth media, in combination with conditioned media (CM) from CA-MSCs and CM from CAFs. Increased tumor cell numbers when co-cultured with CA-MSC (A) or CA-MSC CM (A) demonstrate the ability of CA-MSCs to induce proliferation in the cancer cells. *p

Effect of combination treatment on COX-2 and Pdcd4 expression

Related Article from Metformin and Cancer Stem Cells: Old Drug, New Targets

Impact of drug concentration and kinase inhibitor selection on synergistic response (L3.6pl model)

Supplementary Figure 7. A. Sections from orthotopic pancreatic tumors from control shRNA- MSC vs GM-CSF-shRNA-MSC were analyzed for expression of F4/80, and Arg1 by immunohistochemical and immunofluorescence staining. B. Quantitation of F4/80 positively labeled cells was performed by light microscopy at an optical magnification of 400Ã-, (n=5 animals per group *p

XLSX file - 13K, shRNA, siRNA, and qPCR primer sequences used.

Supplementary Figure 5. Representative Western blot demonstrating the expression of GM-CSF receptors CSF2Rα and CSF2Rβ in primary human pancreatic cancer cells.

Supplementary Methods Supplemental Figure 1: The MIA-PaCa-2 + PCAF model exhibits histologic features consistent with desmoplasia 90 days post implantation Supplemental Figure 2: 1 μM calcipitriol activates the vitamin D receptor (VDR) in cancer associated fibroblasts Supplemental Figure 3: VDAR activation + PDP enable a 75% dose reduction in nal-IRI Supplemental Figure 4: Inhibition of the CXCL12/CXCR7 by CAL+PDP is lost by day 90 post-implantation Supplemental Figure 5: Calcipotriol and photodynamic priming cytotoxicity curves Supplemental Figure 6: MRC-5 conditioned medium does not affect the baseline metabolic activity of PDAC cell lines Supplemental Figure 7: PDP increases intratumoral nal-IRI via increased vascular and parenchymal permeability Supplemental Figure 8: Treatment with CAL+PDP+5 mg/kg nal-IRI induces global transcriptomic shifts in pancreataic tumors that correlate with a less aggressive and more responsive treatment profile compared to treatment with PDP+20 mg/kg nal-IRI

PDF file - 1133K, This file contains supplemental figures validating RNA-Seq method for measuring gene expression and nominating outlier kinases in individual cancer samples.

In this issue of the journal, Bao and colleagues report (beginning on page 355) that the antidiabetic drug metformin targets pancreatic cancer stem cells through, at least partially, the modulation of miRNA expression and subsequent regulation of stem cell renewal and signaling factors. In this Perspective, we briefly discuss the cancer stem cell hypothesis, its clinical relevance, and how targeting the mTOR pathway may yield an avenue for disrupting the cancer stem cell compartment and thus yield long-term therapeutic benefit in multiple cancers. Cancer Prev Res; 5(3); 351–4. ©2012 AACR.

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Effect of combination treatment on growth and protein expression in models eliciting high and low synergy scores

CD44 regulates EMT genes.

Supplementary Figure 6. Pancreatic CA-MSCs promote tumor sphere formation. GFP-labeled tumor cells were grown in growth media, in combination with CM from CA-MSCs and CM from CAFs or co-cultured with CA-MSCs or CAFs (red). Enhanced size of tumor spheres when tumor cells were grown with CA-MSCs in sphere forming media (*p

Table S1: Bliss Index for Effect of MEK-I/SHP099 Combination on Cancer Cell Proliferation Table S2: Primer Sequences for qRT-PCR

Evaluation of in vivo efficacy to the combination of trametinib and palbociclib in L3.6pl tumor-bearing animals

Pancreatic cancer is one of the deadliest human malignancies due to its early metastatic spread and resistance to therapy. The mechanisms regulating pancreatic cancer metastasis are so far poorly understood. Here, using both in vitro and in vivo approaches, it is demonstrated that CD44, a transmembrane glycoprotein expressed on a subset of pancreatic cancer cells, is required for the induction of epithelial–mesenchymal transition (EMT) and the activation of an invasive program in pancreatic cancer. Mechanistically, the transcription factor Snail1 (SNAI1), a regulator of the EMT program, is a downstream target of CD44 in primary pancreatic cancer cells and regulates membrane bound metalloproteinase (MMP14/MT1-MMP) expression. In turn, MT1-MMP expression is required for pancreatic cancer invasion. Thus, these data establish the CD44–Snail–MMP axis as a key regulator of the EMT program and of invasion in pancreatic cancer.Implications: This study sets the stage for CD44 and MT1-MMP as therapeutic targets in pancreatic cancer, for which small molecule or biologic inhibitors are available.Visual Overview: http://mcr.aacrjournals.org/content/early/2014/09/10/1541-7786.MCR-14-0076/F1.large.jpg.Mol Cancer Res; 13(1); 9–15. ©2014 AACR.

Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity.

Supplementary Figure 1. A. FACS analysis of freshly digested pancreatic tumor tissue from two different PDA patients (left) and outgrown CAFs from matching patients cultured using the outgrowth method for 2 weeks (right), demonstrating the percentage of CA-MSCs expressing CD44+ CD73+ CD90+ CD49α+ markers. B. ELISA data quantifying amount of GM-CSF secreted by freshly isolated CA-MSCs and cultured CA-MSCs. C. Sorted single CA-MSCs demonstrate multipotent differentiation capacity in differential culture conditions. Specific cell stains used were Alizarin Red for bone, Oil Red O for adipose and Alcian Blue for cartilage.

The ineffectiveness of chemotherapy in patients with pancreatic cancer highlights a critical unmet need in pancreatic cancer therapy. Two commonly mutated genes in pancreatic cancer, KRAS and CDKN2A, have an incidence exceeding 90%, supporting investigation of dual targeting of MEK and CDK4/6 as a potential therapeutic strategy for this patient population. An in vitro proliferation synergy screen was conducted to evaluate response of a panel of high passage and patient-derived pancreatic cancer models to the combination of trametinib and palbociclib to inhibit MEK and CDK4/6, respectively. Two adenosquamous carcinoma models, L3.6pl and UM59, stood out for their high synergy response. In vivo studies confirmed that this combination treatment approach was highly effective in subcutaneously implanted L3.6pl and UM59 tumor-bearing animals. Both models were refractory to single-agent treatment. Reverse-phase protein array analysis of L3.6pl tumors excised from treated animals revealed strong downregulation of COX-2 expression in response to combination treatment. Expression of COX-2 under a CMV-driven promoter and shRNA knockdown of COX-2 both led to resistance to combination treatment. Our findings suggest that COX-2 may be involved in the improved therapeutic outcome seen in some pancreatic tumors that fail to respond to MEK or CDK4/6 inhibitors alone but respond favorably to their combination.

Supplementary Figure S1: Combined SHP2/MEK inhibition prevents adaptive resistance in PDAC and NSCLC lines Supplementary Figure S2: SHP2 inhibition abrogates MEK-I-evoked reactivation of the ERK MAPK pathway Supplementary Figure S3: Distinct mechanisms of resistance to MEK-I/SHP099 combination in PDAC lines Supplementary Figure S4: Development of tolerable SHP099/trametinib regimen Supplementary Figure S5: Combination decreases tumor cell proliferation and promotes programmed cell death (PDAC and NSCLC) Supplementary Figure S6: Combination decreases tumor cell proliferation and promotes programmed cell death (PDAC, TNBC and HGSC) Supplementary Figure S7: RTKs/RTK ligand expression and Annexin V/cell cycle analysis of TNBC and HGSC lines

XLSX file - 2238K, Complete kinome compendium of all tissue types by RNA-Seq.

Supplementary Figure 4. Differential secretion of cytokines by CA-MSC and CAFs from four different patients.

Detailed Supplementary Method

Supplementary Figure 3. Pancreatic CA-MSCs promote tumor growth. A. BXPC-3 (wild type KRAS) pancreatic cancer cells (5 x 104) transduced with a luciferase-expressing lentivirus were orthotopically injected alone or with same number of CAF or CA-MSC into the pancreatic tail of NOD/SCID mice (n=4 per group). Representative bioluminescent images of animals in each group are shown at 4 weeks after injection depicting tumor growth. B. Bioluminescent-based tumor growth (measured by signal intensity p/s/cm2) of luciferase tumors alone or in combination with CAFs or CA-MSCs (MSCs). Pooled results of the average tumor size {plus minus} SEM from each group. *p

PDF file - 4323K, This file contains supplemental figures providing results of additional experiments supporting sample-specific sensitivities upon targeting of outlier kinases.

Purpose: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy.Experimental Design: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays.Results: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition.Conclusions: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling. Clin Cancer Res; 18(19); 5374–86. ©2012 AACR.

PDF file - 173K, Association of the HAb18G/CD147 expression with, and correlation between HAb18G/CD147-CD44s-pSTAT3 co-expression and the clinicopathologic factors (related to Figure 5B-C).

Purpose: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models.Experimental Design: We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1.Results: AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism.Conclusions: AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1. Clin Cancer Res; 17(11); 3706–15. ©2011 AACR.

PDF file - 451K, Figure S5: CyPA promotes HAb18G/CD147-CD44s-pSTAT3 signaling and correlated pancreatic cell growth (related to Figure 2, 3). Figure S6: HAb18G/CD147 and CD44s are co-expressed and associated in pancreatic cancer (related to Figure 4).

Supplementary Figure 1 Legend. Median overall survival correlated with change in CA 19-9 (>50% decrease, versus {less than or equal to}50% decrease, or primary increase)

Supplementary Figure 1. Median overall survival correlated with change in CA 19-9 (>50% decrease, versus {less than or equal to}50% decrease, or primary increase) compared to baseline.

PDF file - 263K, The effects of HAb18G/CD147 promoting pancreatic tumor development are confirmed with pGIPZ and pTRIPZ CD147 shRNA (related to Figure 1).

PDF file - 147K, Oncomine data analysis of CD147 expression in pancreatic cancer and its co-expression genes (related to the 7th paragraph of Introduction).

PDF file - 61K, The efficiency of HAb18G/CD147 knock-down using pGIPZ and pTRIPZ shRNA vector in human pancreatic cancer cells (related to Figure 1).