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Single-cell transcriptomics has emerged as the preferred tool to define cell identity through the analysis of gene expression signatures. However, there are limited studies that have comprehensively compared the performance of different scRNAseq systems in complex tissues. Here, we present a systematic comparison of three well-established high throughput 3’-scRNAseq platforms: Drop-seq, 10x Chromium and BD Rhapsody; using tumours that present high cell diversity. Our experimental design includes both fresh and artificially damaged samples from the same tumours, which also provides a comparable dataset to examine their performance under challenging conditions. The performance metrics used in this study consist of gene sensitivity, mitochondrial content, reproducibility, clustering capabilities, cell type representation and ambient RNA contamination. These analyses showed that BD Rhapsody and 10x Chromium have similar but higher gene sensitivity than Drop-seq, while BD Rhapsody has the highest mitochondrial content. Interestingly, we found cell type detection biases between platforms, including a lower proportion of endothelial and myofibroblast cells in BD Rhapsody and lower gene sensitivity in granulocytes for 10x Chromium. Moreover, the source of the ambient noise was different between plate-based and droplet-based platforms. In conclusion, our reported platform differential performance should be considered for the selection of the scRNAseq method during the study experimental designs.

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084–94. ©2016 AACR.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.Significance:This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

miR-139-5p overexpression significantly increased radiation sensitivity in 10 BC cell lines (A-I), and also sensitivity to ROS generator menadione (J).

Purpose: To develop effective combination therapy against pancreatic ductal adenocarcinoma (PDAC) with a combination of chemotherapy, CHK1 inhibition, and EGFR-targeted radioimmunotherapy.Experimental Design: Maximum tolerated doses were determined for the combination of gemcitabine, the CHK1 inhibitor PF-477736, and Lutetium-177 (177Lu)–labeled anti-EGFR antibody. This triple combination therapy was investigated using PDAC models from well-established cell lines, recently established patient-derived cell lines, and fresh patient-derived xenografts. Tumors were investigated for the accumulation of 177Lu-anti-EGFR antibody, survival of tumor-initiating cells, induction of DNA damage, cell death, and tumor tissue degeneration.Results: The combination of gemcitabine and CHK1 inhibitor PF-477736 with 177Lu-anti-EGFR antibody was tolerated in mice. This triplet was effective in established tumors and prevented the recurrence of PDAC in four cell line–derived and one patient-derived xenograft model. This exquisite response was associated with the loss of tumor-initiating cells as measured by flow cytometric analysis and secondary implantation of tumors from treated mice into treatment-naïve mice. Extensive DNA damage, apoptosis, and tumor degeneration were detected in the patient-derived xenograft. Mechanistically, we observed CDC25A stabilization as a result of CHK1 inhibition with consequent inhibition of gemcitabine-induced S-phase arrest as well as a decrease in canonical (ERK1/2 phosphorylation) and noncanonical EGFR signaling (RAD51 degradation) as a result of EGFR inhibition.Conclusions: Our study developed an effective combination therapy against PDAC that has potential in the treatment of PDAC. Clin Cancer Res; 20(12); 3187–97. ©2014 AACR.

SLC7A11 knockdown in PDAC CAFs does not affect glutamate secretion and is maintained in the presence of oxidative stress.

Supplementary Figure 1: Sensitization of PANC-1 cells to gemcitabine and EGFR-directed RIT by Chk1 inhibition. (A) Adherent cultures of the PDAC PANC-1 cell line were treated in the absence or presence of escalating doses of gemcitabine, in the absence of presence of Chk1i alone (PF-477736, 180 nM) or the combinations. Chk1i was added either 16 hours after gemcitabine or concurrently. Cells were collected 24-96 hours after treatment for standard cell cycle analysis by DNA content using FACS. (B) PANC-1 cells were treated with escalating concentrations of 177Lu-anti-EGFR mAb (RIT) to achieve the specified radiation doses (0-4 Gy) over 72 hours of incubation. RIT was performed alone or in combination with Chk1i (180 nM) and then clonogenic survival was determined by standard assays. Chk1i alone at 180 nM did not have any effect on clonogenic survival (data not shown). (C) PANC-1 cells were left untreated (vehicle control, data not shown) or incubated with anti-EGFR mAb (unlabeled, control which was not different from the vehicle control), 177Lu-DOTA, 177Lu-labelled mAb with irrelevant specificity (Sal5, raised against Salmonella antigen) or 177Lu-anti-EGFR mAb (2 Gy over 72 hours). Cells were washed 3 hours after incubation to remove unbound material and left untreated or treated with Chk1i alone (180 nM), gemcitabine alone (40 ng/mL) or the combination (Chk1i+gemcitabine) before standard clonogenic survival assays.

Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.

Supplementary Figure 3: EGFR expression in the PANC-1 and BxPC-3. Cell cultures were used for standard immunoblot and flow cytometry analysis for EGFR expression. The anti-EGFR mAb clone 225 was used for staining and anti-mouse IgG antibody conjugated with HRP was used for immunoblot or conjugated with Alexa488 for flow cytometry. Anti-tubulin was used to confirm equal loading for immunoblot. Both PDAC cell lines express EGFR.

Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilution of RNA concentration was used to establish a standard curve for determining the reaction efficiency of PARP1, PARP2, PARP3, and housekeeper gene (PPIA: cyclophilin A) TaqMan RT-PCR assays in both rat and mouse species; Determination of optimal reference gene for rat-mouse data normalization. Suppl Fig. 7.A. A serial dilution of reference RNA (Qiagen) concentration was used to establish a standard curve for determining the reaction efficiency of PARP3 and housekeeper genes (PPIA: cyclophilin and YWHAZ: tyrosine 3-monooxygenase/tryptohpan 5-monooxygenase activation protein, zeta polypeptide) SybrGreen RT-PCR assays in both rat and human species; Determination of optimal reference gene for rat-human data normalization. Suppl Fig. 8. Relative body weight changes in rat following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide; Relative body weight changes in rat following treatment with 20 mg/kg AZD2461 and olaparib in combination with temozolomide.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321–36. ©2018 AACR.

Supplementary Figure 2: Maximum tolerated doses of gemcitabine, Chk1i and EGFR-directed RIT in vivo. (A) Balb/c nude mice bearing PANC-1 subcutaneous xenografts (60 mm3 in volume) were treated with 50 mg/kg or 100 mg/kg of gemcitabine on days 1, 4, 7 and 10 administered intravenously combined with 15 mg/kg Chk1i, administered as two doses per day (7.5 mg/kg per dose) on days 1, 4, 7 and 10 at 3 hours before and after gemcitabine administration. Mice were treated with gemcitabine and Chk1i combinations (gem + Chk1i) alone or in combination with 6 or 9 MBq/20g (300 or 450 MBq/kg) of 177Lu-anti-EGFR mAb. Mice were monitored for 14 days to determine acute toxicities as judged by weight, posture, movement, eating and drinking. Tumor volume was also monitored during this short time as an indication of efficacy. (B) The tumor growth curves were used to measure the growth rate (k = day-1) from exponential growth equations and shown in the bar graph in panel (5 mice per group, error bar is the standard error of the mean, SEM). *** p < 0.001 in One-way ANOVA in GraphPad® Prism comparing treatments involving anti-EGFR RIT and those not involving this RIT (p

Detailed methods of: EGFR immunohistochemistry of PDAC tissue microarrays Patient derived xenografts (PDXs) and cell lines (PDCLs) generation Cell culture Treatments in vitro Histological and immunoblotting analyses of PDXs post-treatment

Supplemental Figure S1. No effect of ROCK inhibitors on the expression of ROCK1 or ROCK2. Supplemental Figure S2. ROCK1 and ROCK2 knockdown, dose-response relationships and IC50 determinations for AT13148 on cell morphology parameters. Supplemental Figure S3. Dose-response relationships for AT13148 on nuclear morphology parameters. Supplemental Figure S4. No effect of ROCK inhibitor treatment on mouse weights. Supplemental Figure S5. AT13148 blocks PDAC tumor growth in vivo.

SLC7A11 expression in iCAFs, myCAFs and quiescent PSCs.

Supplementary Tables 1-6 Supp. Table 1: Clinicopathological characteristics of breast cancer patients whose tumours were profiled by miRNA expression microarrays to identify miRNAs associated with local relapse following radiotherapy. Supp. Table 2: Clinicopathological characteristics of RT-treated breast cancer patients from "Rotterdam", "Uppsala", and "Mainz" cohorts that were analysed for biomarker validation Supp. Table 3: Clinicopathological characteristics of RT- and non-RT treated breast cancer patients from "Lund" cohort that were analysed for biomarker validation Supp. Table 4: Clinicopathological characteristics of RT-treated breast cancer patients from "Oxford" and "METABRIC" cohorts that were analysed to validate miR-139-5p as a biomarker Supp. Table 5: Primer sequences for miR-139-5p targets (Roche Universal Probe Library QPCR system) Supp. Table 6: IC50s for 15 DNA damaging compounds in 10 breast cancer cell lines. Also listed are DNA mutations significantly correlated (p

List of antibodies

Figure S1: Treatment response to cisplatin spread over several days. Figure S2: Cisplatin adducts in tumor remants; GFP-marker in the BRCA1-deficient mouse model. Figure S3: EdU incorporation assay. Figure S4: Genomic profile and clonogenic assays of BRCA1-deficient cell lines. Figure S5: Determination of IC50 in KB1P-B11 and -G3; Cell cycle dependent cell death of KB1P-B11-FUCCI. Figure S6: Drug response to nimustine, melphalan, bendamustine or temozolomide. Figure S7: High dose treatment of mice with carboplatin or thiotepa. Figure S8: Combination or single treatment with cyclophosphamide, carboplatin or thiotepa.

Radiotherapy is essential to the treatment of most solid tumors and acquired or innate resistance to this therapeutic modality is a major clinical problem. Here we show that miR-139-5p is a potent modulator of radiotherapy response in breast cancer via its regulation of genes involved in multiple DNA repair and reactive oxygen species defense pathways. Treatment of breast cancer cells with a miR-139-5p mimic strongly synergized with radiation both in vitro and in vivo, resulting in significantly increased oxidative stress, accumulation of unrepaired DNA damage, and induction of apoptosis. Several miR-139-5p target genes were also strongly predictive of outcome in radiotherapy-treated patients across multiple independent breast cancer cohorts. These prognostically relevant miR-139-5p target genes were used as companion biomarkers to identify radioresistant breast cancer xenografts highly amenable to sensitization by cotreatment with a miR-139-5p mimetic.Significance: The microRNA described in this study offers a potentially useful predictive biomarker of radiosensitivity in solid tumors and a generally applicable druggable target for tumor radiosensitization. Cancer Res; 78(2); 501–15. ©2017 AACR.

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Supplementary Tables 1-4 from Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy. Cancer Res; 72(9); 2350–61. ©2012 AACR.

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Supplementary Figures 1-4 from Moderate Increase in Mdr1a/1b Expression Causes In vivo Resistance to Doxorubicin in a Mouse Model for Hereditary Breast Cancer

Supplementary Tables 1-3 from Moderate Increase in Mdr1a/1b Expression Causes In vivo Resistance to Doxorubicin in a Mouse Model for Hereditary Breast Cancer

Supplementary Figure Legends 1-4 from Moderate Increase in Mdr1a/1b Expression Causes In vivo Resistance to Doxorubicin in a Mouse Model for Hereditary Breast Cancer

Supplementary Materials and Methods from Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

Supplementary Table 1, Supplementary Figures 1-5 from Small-Molecule Multidrug Resistance–Associated Protein 1 Inhibitor Reversan Increases the Therapeutic Index of Chemotherapy in Mouse Models of Neuroblastoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene “TLR2 activation” signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.

How cancer cells adapt to stress Bacteria adapt to harsh conditions such as antibiotic exposure by acquiring new mutations, a process called stress-induced mutagenesis. Cipponi et al. investigated whether similar programs of mutagenesis play a role in the response of cancer cells to targeted therapies. Using in vitro models of intense drug selection and genome-wide functional screens, the authors found evidence for an analogous process in cancer and showed that it is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. This pathway appears to mediate a stress-related switch to error-prone DNA repair, resulting in the generation of mutations that facilitate the emergence of drug resistance. Science , this issue p. 1127

Pancreatic acinar cell carcinoma is relatively rare (1 to 2% of pancreatic malignancies) but may be under-recognized. In contrast to pancreatic ductal adenocarcinoma, most acinar cell carcinomas lack mutations in KRAS, DPC, CDKN2A or TP53, but appear to have a high incidence of gene rearrangements, with up to 20% reported to be driven by BRAF fusions. With the development of a new class of RET-specific tyrosine kinase inhibitors, which appear to have particularly strong activity against RET gene rearranged tumours, there is now considerable interest in identifying RET gene rearrangements across a wide range of cancers. RET rearrangements have been reported to occur at a very low incidence (