Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

Delta Radiomic Features Predict Resection Margin Status and Overall Survival in Neoadjuvant-Treated Pancreatic Cancer Patients.

BACKGROUND: Neoadjuvant therapy (NAT) emerged as the standard of care for patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgery; however, surgery is morbid, and tools to predict resection margin status (RMS) and prognosis in the preoperative setting are needed. Radiomic models, specifically delta radiomic features (DRFs), may provide insight into treatment dynamics to improve preoperative predictions. METHODS: We retrospectively collected clinical, pathological, and surgical data (patients with resectable, borderline, locally advanced, and metastatic disease), and pre/post-NAT contrast-enhanced computed tomography (CT) scans from PDAC patients at the University of Texas Southwestern Medical Center (UTSW; discovery) and Humanitas Hospital (validation cohort). Gross tumor volume was contoured from CT scans, and 257 radiomics features were extracted. DRFs were calculated by direct subtraction of pre/post-NAT radiomic features. Cox proportional models and binary prediction models, including/excluding clinical variables, were constructed to predict overall survival (OS), disease-free survival (DFS), and RMS. RESULTS: The discovery and validation cohorts comprised 58 and 31 patients, respectively. Both cohorts had similar clinical characteristics, apart from differences in NAT (FOLFIRINOX vs. gemcitabine/nab-paclitaxel; p < 0.05) and type of surgery resections (pancreatoduodenectomy, distal or total pancreatectomy; p < 0.05). The model that combined clinical variables (pre-NAT carbohydrate antigen (CA) 19-9, the change in CA19-9 after NAT (∆CA19-9), and resectability status) and DRFs outperformed the clinical feature-based models and other radiomics feature-based models in predicting OS (UTSW: 0.73; Humanitas: 0.66), DFS (UTSW: 0.75; Humanitas: 0.64), and RMS (UTSW 0.73; Humanitas: 0.69). CONCLUSIONS: Our externally validated, predictive/prognostic delta-radiomics models, which incorporate clinical variables, show promise in predicting the risk of predicting RMS in NAT-treated PDAC patients and their OS or DFS.

Variant calling enhances the identification of cancer cells in single-cell RNA sequencing data.

Single-cell RNA-sequencing is an invaluable research tool that allows for the investigation of gene expression in heterogeneous cancer cell populations in ways that bulk RNA-seq cannot. However, normal (i.e., non tumor) cells in cancer samples have the potential to confound the downstream analysis of single-cell RNA-seq data. Existing methods for identifying cancer and normal cells include copy number variation inference, marker-gene expression analysis, and expression-based clustering. This work aims to extend the existing approaches for identifying cancer cells in single-cell RNA-seq samples by incorporating variant calling and the identification of putative driver alterations. We found that putative driver alterations can be detected in single-cell RNA-seq data obtained with full-length transcript technologies and noticed that a subset of cells in tumor samples are enriched for putative driver alterations as compared to normal cells. Furthermore, we show that the number of putative driver alterations and inferred copy number variation are not correlated in all samples. Taken together, our findings suggest that augmenting existing cancer-cell filtering methods with variant calling and analysis can increase the number of tumor cells that can be confidently included in downstream analyses of single-cell full-length transcript RNA-seq datasets.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma.

Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway.

Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP(+) ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.

Phosphorylated Histone H3 (PHH3) Is a Superior Proliferation Marker for Prognosis of Pancreatic Neuroendocrine Tumors.

BACKGROUND: The staging of pancreatic neuroendocrine tumors (PNETs) is continuously evolving. Mitotic count, as measured by hematoxylin and eosin (H&E) or Ki67 labeling index (Ki67LI), is the best predictor of disease biology. However, both of these methods have several limitations. Phosphorylated histone H3 (PHH3), a novel mitotic marker, is potentially more accurate and easier to evaluate. This study aimed to evaluate the prognostic impact of PHH3 on patients with PNETs. METHODS: Clinicopathologic data and paraffin-embedded tissue were evaluated for 100 of the 247 PNET patients whose tumors were resected between 1998 and 2010. Mitotic counts were analyzed on H&E-, Ki67-, and PHH3-stained slides by two independent pathologists. Kaplan-Meier curves, log-rank tests, Cox regression models, and time-dependent receiver operative characteristics (ROC) curves were used to evaluate the prognostic power of these markers. An internal data cross-validation was performed to select the best cutoff. RESULTS: Of the 100 PNET patients resected, 53 were men. The median age of the patients was 59 years (range 19-96 years). The median follow-up period was 68 months (range 3-186 months). The median time for evaluation of an H&E- or PHH3-stained slide was 3 min, relative to 15 min for Ki67. The findings showed H&E, Ki67, and PHH3 all to be excellent predictors of disease-specific survival (DSS). However, PHH3 was superior to H&E and Ki67 in predicting both disease-free survival (DFS) (p = 0.006) and DSS (p = 0.001). Evaluation of the PHH3 mitotic count showed 7 mitoses per 10 high-power fields (HPFs) to be the optimal cutoff for differentiating between low- and high-risk PNET patients. CONCLUSIONS: PHH3 is a better predictor of both DFS and DSS than H&E or Ki67 in PNET. In addition, PHH3 appears to be both easier to interpret and more accurate when compared to current prognostic markers.

Ablative Radiation Therapy in Oligometastatic Pancreatic Cancer to Delay Polyprogression, Limit Chemotherapy, and Improve Outcomes.

PURPOSE: The oligometastatic state is observed in patients across many malignancies, with increased recognition regarding improved outcomes after local therapies. However, there is limited data specifically regarding pancreatic ductal adenocarcinoma. We hypothesized that an oligometastatic pancreatic ductal adenocarcinoma (OPanc) phenotype would benefit from stereotactic ablative radiation therapy (SABR) to all active metastatic sites. Here, we report our institutional experience of SABR-treated OPanc to evaluate the feasibility of the approach. METHODS AND MATERIALS: A retrospective review of patients with synchronous or metachronous OPanc (1 to 5 metastases) who received SABR to all active metastatic sites was performed. We identified a comparable group of patients with similar metastatic burden, range of CA19-9 levels, and no progression for at least 5 months who did not receive SABR. We compared overall survival as the primary outcome, and polyprogression-free survival and time off chemotherapy as the secondary exploratory assessments. A third group presenting with stage IV pancreatic ductal adenocarcinoma and more than 5 distant lesions (polymetastatic) was identified to help define expected outcomes after polyprogression. RESULTS: Our study included 20 patients with OPanc receiving SABR and 21 who did not. SABR was delivered to 38 metastatic tumors. Out of the 20 SABR-treated OPanc patients, 17 (85%) had 6 or more months of time off chemotherapy, compared with 7 patients (33.3%) among the chemotherapy-treated group. Median polyprogression-free survival was 40 and 14 months (hazard ratio = 0.2; 95% confidence interval, 0.07-0.54; P = .0009), and overall survival was 42 and 18 months (hazard ratio = 0.21; 95% confidence interval, 0.08-0.53; P = .0003), for SABR- and chemotherapy-treated cohorts, respectively. CONCLUSIONS: Management of OPanc with SABR as local regional therapy could improve outcomes in a selected population and warrants prospective evaluation.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

Mutagens interfere with microRNA maturation by inhibiting DICER. An in silico biology analysis.

Exposure to environmental mutagens results in alteration of microRNA expression mainly oriented towards down-regulation, as typically observed in cigarette smoke. However, the molecular mechanism triggering this event is still unknown. To shed light on this issue, we developed an 'in silico' analysis testing 25 established environmental mutagens (polycyclic aromatic hydrocarbons, heterocyclic compounds, nitrosoamines, morpholine, ethylnitrosurea, benzene derivatives, hydroxyl amines, alkenes) for their potential to interfere with the function of DICER, the enzyme involved in the cytoplasmic phase of microRNA maturation. In order to analyse the binding affinity between DICER and each mutagen, the three-dimensional bioinformatic structures of DICER-RNase III domains and of mutagens have been constructed. The binding affinity of mutagens for each DICER's RNase III domain was estimated by calculating the global contact-energy and the number of intermolecular contacts. These two parameters reflect the stability of the DICER-mutagen complexes. All the 25 mutagens tested form stable complexes with DICER, 20 of which form a complex with DICER A domain, that is more stable than those formed by DICER with its natural substrate, i.e. double strand short RNAs. These mutagens are benzo(a)pyrene diol epoxide, nitroimidazoles, fluorenes, naphthalene, morpholine, stilbenes, hydroxylamines, fecapentenes. In the case of exposure to mutagen mixtures (benzo(a)pyrene-diolepoxide and 4-acetylaminostilbene), synergistic or less than addictive effects occur depending on the docking order of the compounds. A group of 8 mutagens with the highest ability to interfere with this DICER function, was identified by hierarchical cluster analysis. This group included 1-ethyl-1-nitrosourea and 4-nitrosomorpholine. Herein, presented data support the view that mutagens interfere with microRNA maturation by binding DICER. This finding sheds light on a new epigenetic mechanism exerted by environmental mutagens in inducing cell damage.

Cancer-Associated Fibroblasts: Versatile Players in the Tumor Microenvironment.

Cancer-associated fibroblasts (CAFs) are indispensable architects of the tumor microenvironment. They perform the essential functions of extracellular matrix deposition, stromal remodeling, tumor vasculature modulation, modification of tumor metabolism, and participation in crosstalk between cancer and immune cells. In this review, we discuss our current understanding of the principal differences between normal fibroblasts and CAFs, the origin of CAFs, their functions, and ultimately, highlight the intimate connection of CAFs to virtually all of the hallmarks of cancer. We address the remarkable degree of functional diversity and phenotypic plasticity displayed by CAFs and strive to stratify CAF biology among different tumor types into practical functional groups. Finally, we summarize the status of recent and ongoing trials of CAF-directed therapies and contend that the paucity of trials resulting in Food and Drug Administration (FDA) approvals thus far is a consequence of the failure to identify targets exclusive of pro-tumorigenic CAF phenotypes that are mechanistically linked to specific CAF functions. We believe that the development of a unified CAF nomenclature, the standardization of functional assays to assess the loss-of-function of CAF properties, and the establishment of rigorous definitions of CAF subpopulations and their mechanistic functions in cancer progression will be crucial to fully realize the promise of CAF-targeted therapies.

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target.

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.

Neoplastic-Stromal Cell Cross-talk Regulates Matrisome Expression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic reaction, warranting intense cancer-stroma communication. In this study, we interrogated the contribution of the BET family of chromatin adaptors to the cross-talk between PDAC cells and the tumor stroma. Short-term treatment of orthotopic xenograft tumors with CPI203, a small-molecule inhibitor of BET proteins, resulted in broad changes in the expression of genes encoding components of the extracellular matrix (matrisome) in both cancer and stromal cells. Remarkably, more than half of matrisome genes were expressed by cancer cells. In vitro cocultures of PDAC cells and cancer-associated fibroblasts (CAF) demonstrated that matrisome expression was regulated by BET-dependent cancer-CAF cross-talk. Disrupting this cross-talk in vivo resulted in diminished growth of orthotopic patient-derived xenograft tumors, reduced proliferation of cancer cells, and changes in collagen structure consistent with that of patients who experienced better survival. Examination of matrisome gene expression in publicly available data sets of 573 PDAC tumors identified a 65-gene signature that was able to distinguish long- and short-term PDAC survivors. Importantly, the expression of genes predictive of short-term survival was diminished in the cancer cells of orthotopic xenograft tumors of mice treated with CPI203. Taken together, these results demonstrate that inhibiting the activity BET proteins results in transcriptional and structural differences in the matrisome are associated with better patient survival. IMPLICATIONS: These studies highlight the biological relevance of the matrisome program in PDAC and suggest targeting of epigenetically driven tumor-stroma cross-talk as a potential therapeutic avenue.

The impact of stapled transanal rectal resection on anorectal function in patients with obstructed defecation syndrome.

PURPOSE: A careful preoperative selection of patients was performed in order to identify those eligible for stapled transanal rectal resection to correct obstructed defecation syndrome. The aim was to assess the consequences of surgery on anorectal function and patient outcomes. METHODS: From January 2004 to June 2007, 33 female patients (median age, 56.3 years; range, 27-77 years) eligible for stapled transanal rectal resection completed standardized questionnaires for the assessment of constipation (constipation scoring system), quality of life (Patient Assessment of Constipation-Quality of Life Questionnaire), and patient satisfaction (visual analogue scale). A complete clinical reassessment including anorectal manometry and defecography was performed after one year. RESULTS: At a median follow-up of 18 months, significant improvement in constipation scoring system, quality of life, and visual analog scale (P < 0.0001) was observed. Postoperative defecography confirmed the correction of internal rectal prolapse (P < 0.01) and rectocele (P < 0.0001) with an increase in rectal sensitivity (P < 0.0001). Significant correlations were observed between rectocele correction and rectal sensitivity, as evidenced by a decrease in rectal sensory threshold volumes (P = 0.017; Phi = 0.7), increased rectal sensitivity, and patient's satisfaction index (P = 0.011; Phi = 0.64). CONCLUSIONS: Stapled transanal rectal resection allowed for the correction of rectocele and intussusceptions. These corrections increased rectal sensitivity, diminished symptoms of obstructed defecation syndrome, and improved the quality of life of patients.

Clinical applications of autologous cryoplatelet gel for the reconstruction of the maxillary sinus. A new approach for the treatment of chronic oro-sinusal fistula.

The authors report their clinical experience regarding an original method of surgical repair of oro-sinusal communications. From September 1999 to December 2003, 13 patients (7 male and 6 female patients; mean age: 52 years, range: 24-68 years) underwent surgical repair of an oro-antral fistula by means of cryoplatelet gel: in three patients, it was mixed with bioglass granules; in two, it was mixed with Bioss; in three, it was mixed with particulate bone extracted by means of a bone grafter from the oral cavity close to the operative site, with addition of demineralised bovine bone; in three, it was used together with porose hydroxyapatite, and in two patients the cryoplatelet gel was used only. No postoperative complication was reported; primary wound healing was achieved within seven to nine days. A bony orthopantoscintigraphy was performed a few months following the operative procedure, showing an active osteogenic process. In eight patients, a CT was performed after 8 to 12 months from the operation, showing a normal pneumatization with reconstruction of the floor of the maxillary sinus. Although preliminary, these findings seem to suggest that the use of bioengineered materials coupled with growth factors and osteoprogenitor cells may represent a valuable alternative to autologous bone transplantation for the reconstruction of the maxillary sinus.

Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.

Growing evidence suggests that microbes can influence the efficacy of cancer therapies. By studying colon cancer models, we found that bacteria can metabolize the chemotherapeutic drug gemcitabine (2',2'-difluorodeoxycytidine) into its inactive form, 2',2'-difluorodeoxyuridine. Metabolism was dependent on the expression of a long isoform of the bacterial enzyme cytidine deaminase (CDDL), seen primarily in Gammaproteobacteria. In a colon cancer mouse model, gemcitabine resistance was induced by intratumor Gammaproteobacteria, dependent on bacterial CDDL expression, and abrogated by cotreatment with the antibiotic ciprofloxacin. Gemcitabine is commonly used to treat pancreatic ductal adenocarcinoma (PDAC), and we hypothesized that intratumor bacteria might contribute to drug resistance of these tumors. Consistent with this possibility, we found that of the 113 human PDACs that were tested, 86 (76%) were positive for bacteria, mainly Gammaproteobacteria.

A tunable delivery platform to provide local chemotherapy for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and painful cancers. It is often highly resistant to therapy owing to inherent chemoresistance and the desmoplastic response that creates a barrier of fibrous tissue preventing transport of chemotherapeutics into the tumor. The growth of the tumor in pancreatic cancer often leads to invasion of other organs and partial or complete biliary obstruction, inducing intense pain for patients and necessitating tumor resection or repeated stenting. Here, we have developed a delivery device to provide enhanced palliative therapy for pancreatic cancer patients by providing high concentrations of chemotherapeutic compounds locally at the tumor site. This treatment could reduce the need for repeated procedures in advanced PDAC patients to debulk the tumor mass or stent the obstructed bile duct. To facilitate clinical translation, we created the device out of currently approved materials and drugs. We engineered an implantable poly(lactic-co-glycolic)-based biodegradable device that is able to linearly release high doses of chemotherapeutic drugs for up to 60 days. We created five patient-derived PDAC cell lines and tested their sensitivity to approved chemotherapeutic compounds. These in vitro experiments showed that paclitaxel was the most effective single agent across all cell lines. We compared the efficacy of systemic and local paclitaxel therapy on the patient-derived cell lines in an orthotopic xenograft model in mice (PDX). In this model, we found up to a 12-fold increase in suppression of tumor growth by local therapy in comparison to systemic administration and reduce retention into off-target organs. Herein, we highlight the efficacy of a local therapeutic approach to overcome PDAC chemoresistance and reduce the need for repeated interventions and biliary obstruction by preventing local tumor growth. Our results underscore the urgent need for an implantable drug-eluting platform to deliver cytotoxic agents directly within the tumor mass as a novel therapeutic strategy for patients with pancreatic cancer.

Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.