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Suppl. Figure 3. A. Immunoblot of LAMC2 upon Lamc2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines. B. Quantification of organoid number upon LAMC2 knockdown by a specific shRNA in 511950 and PM12167 PDAC mouse cell lines.C. Quantification of organoid area (AU) in same cell lines as in B. Error bars correspond to s.d. D. Immunoblot of LAMC2 upon LAMC2 knockdown in HPAFII cell line. E. Quantification of organoid number and organoid area (AU) in HPAFII cell line expressing a TET-inducible shRNA against LAMC2 (bottom) or a control shRNA (top) cultured in 3D. F. Representative images of the subcutaneous tumors from PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. G. Representative images of the subcutaneous tumors from HPAFII cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. H and I. Representative IHC of LAMC2 expression in subcutaneous tumors from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice J. Representative images of the tumors derived from intrapancreatic injection of PATU8902 cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. K. Representative images of the subcutaneous tumors from PM12167 mouse cells expressing a shRNA against Lamc2 or a control shRNA at sacrifice. L and M. Representative images of the subcutaneous (sc) tumors from PATU8902 and HPAFII expressing a TET-inducible shRNA against LAMC2 or a control shRNA at sacrifice. ShRNA expression induced after tumor establishment at Day 8. N and O. Representative IHC of LAMC2 expression in subcutaneous tumors (Tx) from PATU8902 (H) and HPAFII (I) cells expressing a TET-inducible shRNA against LAMC2 or a control shRNA, shRNA expression was induced at day 8 of tumor establishment. P. Quantification of Ki67 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx. Q. Quantification of cleaved caspase3 IHC staining in PATU8902 and HFAFII xenograft models: SC and Tx.

This file includes the Suppl. Methods, Table (1) and References

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Purpose:The identification of pancreatic ductal adenocarcinoma (PDAC) dysregulated genes may unveil novel molecular targets entering inhibitory strategies. Laminins are emerging as potential targets in PDAC given their role as diagnostic and prognostic markers. Here, we investigated the cellular, functional, and clinical relevance of LAMC2 and its regulated network, with the ultimate goal of identifying potential therapies.Experimental Design:LAMC2 expression was analyzed in PDAC tissues, a panel of human and mouse cell lines, and a genetically engineered mouse model. Genetic perturbation in 2D, 3D, and in vivo allograft and xenograft models was done. Expression profiling of a LAMC2 network was performed by RNA-sequencing, and publicly available gene expression datasets from experimental and clinical studies examined to query its human relevance. Dual inhibition of pharmacologically targetable LAMC2-regulated effectors was investigated.Results:LAMC2 was consistently upregulated in human and mouse experimental models as well as in human PDAC specimens, and associated with tumor grade and survival. LAMC2 inhibition impaired cell cycle, induced apoptosis, and sensitized PDAC to MEK1/2 inhibitors (MEK1/2i). A LAMC2-regulated network was featured in PDAC, including both classical and quasi-mesenchymal subtypes, and contained downstream effectors transcriptionally shared by the KRAS signaling pathway. LAMC2 regulated a functional FOSL1–AXL axis via AKT phosphorylation. Furthermore, genetic LAMC2 or pharmacological AXL inhibition elicited a synergistic antiproliferative effect in combination with MEK1/2is that was consistent across 2D and 3D human and mouse PDAC models, including primary patient-derived organoids.Conclusions:LAMC2 is a molecular target in PDAC that regulates a transcriptional network that unveils a dual drug combination for cancer treatment.

Background High blood pressure (BP) is a major risk factor for cardiovascular disease (CVD). Consumption of dark chocolate, which is high in flavonoids that may reduce CVD risk, is an attractive intervention to reduce to BP. Additionally, the use of mobile health (mHealth) technologies (eg, telehealth, smartphones, and wearable devices) can improve outcomes in patients with CVD. Objective The purpose of this study was to investigate the impact of dark chocolate intake on BP, subject use of mHealth, and integration of mHealth into a clinical trial. Methods The COCOA-BP (ChOcolate COnsumption And Blood Pressure) study was a prospective, single-center, pre-/postintervention study that enrolled 62 healthy volunteers. The study consisted of 3 phases: smartwatch/smart BP monitor familiarization and washout from chocolate (week 1); control (week 2); and intervention (weeks 3 and 4). During the intervention phase, subjects consumed 50 g of dark chocolate per day. The primary endpoint was change in resting systolic BP between the intervention and control phases. Additional endpoints included device accuracy and correlation with physical activity. Results Mean resting systolic BP was 116.4 mm Hg before chocolate intake among 62 participants (mean age 37 years; 61% female). After chocolate intake, mean resting systolic BP was 116.0 mm Hg (difference –0.4; P = .69). These findings suggest that 2 weeks of dark chocolate intake did not reduce resting systolic BP. There was poor agreement between mHealth device and standard (nurse-performed) measurements. Conclusion In this study, short-term dark chocolate intake did not seem to reduce BP. mHealth technology shows great potential for use in clinical studies, but challenges related to device accuracy and compliance need to be addressed.

SummaryOvarian cancer metastasizes into the peritoneum through dissemination of transformed epithelia as multicellular spheroids1, 2. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues3: lumen surrounded by smoothly contoured, adhered, and immotile epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid ‘moruloid’ clusters that permit intercellular movement and penetration by new cells. Moruloid clusters can coalesce to form bigger clusters. Upon further culture, moruloid clusters mature into ‘blastuloid’ spheroids with smooth contours, lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow penetration by new cells. Ultrastructural examination reveals a basement membrane-like matrix coat on the surface of blastuloid, but not moruloid, spheroids: immunocytochemistry confirms the presence of extracellular matrix proteins: Collagen IV and Laminin-322. Enzymatic debridement of the coat results in a reversible loss of lumen and contour. Debridement also allows spheroidal coalescence and cell intrusion in blastuloid spheroids and enhances adhesion to peritoneal substrata. Therefore, the dynamical matrix coat regulates both the morphogenesis of cancer spheroids and their adhesive interaction with their substrata, affecting ultimately the progression of the disease.ResultsSurvival of women afflicted with epithelial ovarian cancer (EOC) trails behind other gynecological malignancies, despite improvements in surgical-pharmacological approaches4,5. The morbidity associated with the disease is a consequence of its transcoelomic route of metastasis: transformed epithelia of the fallopian tubes and ovaries in the form of spheroids, eventually home and adhere to the mesothelial lining of the peritoneum, occasionally invade through the underlying collagenous extracellular matrix and form secondary metastatic foci around abdominal organs1,6, 7. EOC spheroids impede the drainage of the fluid from the peritoneal cavity and alter its composition; in turn the fluid, now known as malignant ascites serves as a pro-tumorigenic milieu for the spheroids8, 9The formation and presence of spheroids within ascites of an ovarian cancer patient is strongly associated with recurrence of cancer and greater resistance to chemotherapy10. Therefore, in order to develop novel strategies to target spheroidal metastatic niche, it is essential to investigate mechanisms that underlie their morphogenesis. Several proteins have been proposed to mediate the adhesion between ovarian cancer epithelia that give rise to spheroids. These include transmembrane receptors such as CD4411, cell adhesion molecules, such as E-cadherin and N-cadherin12, matrix adhesion-inducing proteins such as integrins13, 14. Remarkably, a phase-contrast microscopic examination of spheroids from patients, or from aggregated epithelia of immortalized cancer lines cultured on low attachment substrata, shows features of morphogenetic organization: presence of a central lumen, radially arranged apposed epithelia and compacted surfaces. Such traits are cognate to organized morphogenesis within the glandular epithelial organs,15which are built through principles that include, but are not limited to, cell-cell adhesion16, 17. In fact, loss of tissue architecture seen in tumorigenesis involves the disappearance of such morphogenetic traits (such as matrix adhesion and polarity)18, 19.In this manuscript, we investigate how these traits are recapitulated in a fluid metastatic context. Using spheroids from patients with high grade serous adenocarcinoma and ovarian cancer cell lines, we show that the development of a basement membrane (BM)-like coat of extracellular matrix is responsible for the compaction and stability of cancer spheroids, for decreasing the motility of cells within it and for generation of lumen. The coat, which is rapidly replenished by cells upon enzymatic debridement, also prevents the attachment of spheroids to matrix substrata. This may have significant implications for the build-up of the massive cellular fraction within the malignant ascites of patients afflicted with ovarian cancer.

Background: Pancreatic cancer stands as one of the deadliest tumors, in part due to the limited efficacy of currently existing therapies. Pancreatic cancer is characterized at the genomic level by the almost universal presence of KRAS mutations. Phenotypically, it features an incipient desmoplastic stroma with a protein component formed by collagens, fibronectin and laminins. A member of the laminin family, LAMININ γ2 or LAMC2, was previously identified by our group as part of a cross-tumors KRAS signature whose high expression was a marker of poor survival in PDAC patients. While a role for LAMC2 in migration and invasion has been previously described, little is known about a potential function in cell proliferation and viability. Moreover, LAMC2 inhibition has been shown to enhance the activity of conventional chemotherapeutic agents. However, whether its abrogation can increase the effect of targeted therapies is yet to be defined. Finally, the direct relationship between LAMC2 and the KRAS oncogene network is yet to be defined. Methods: A meta-analysis of several human PDAC data sets and survival analysis of the TCGA data were performed to query the expression levels and prognosis significance of LAMC2. Human and mouse PDAC cell lines as well as a mouse model of PDAC (KrasLSLG12D, Tp53f/f, Ptf1aCre) were used to assess LAMC2 expression. In vitro (2D and 3D organoids) and in vivo models derived from human and mouse PDAC cell lines or primary tumors were used to define the functional role of LAMC2 in PDAC. Cellular and molecular analysis were deployed to dissect the mechanism of action of LAMC2 in PDAC. A dual pharmacological combination based on LAMC2-regulated effectors was also tested in human and mouse pancreatic cancer models. Results: At the clinical level, we observed that LAMC2 is overexpressed in human PDAC patients with regard to normal tissue. The overexpression of LAMC2 was recapitulated in human and mouse cell lines. Genetic depletion of LAMC2 had an adverse effect in 2D proliferation, clonogenic efficiency, 3D organoid growth, and PDAC-based xenograft/allograft tumor development. This deleterious effect was driven by an induction in apoptosis and a decrease in S phase consistent across species. Mechanistically, LAMC2 was linked to the KRAS pathway via transcriptional regulation of the transcription factor FOSL1/AP1, which was also a member of the cross-tumors signature. Furthermore, LAMC2 controlled a gene signature that overlaps with KRAS- and FOSL1-regulated gene signatures, which was also highly expressed in various pancreatic cancer data sets, and includes the targetable kinase AXL. Lastly, concomitant pharmacological inhibition of AXL and the FOSL1 upstream regulator MEK1/2 displayed a more adverse effect on human and pancreatic pancreatic cancer than each single drug alone. Conclusion: These data suggest that LAMC2 is a molecular target tightly linked to KRAS oncogene signaling that regulates downstream effectors amenable to the development of combination therapies. Citation Format: Shruthi Narayanan, Oihane Erice, Iker Feliu, Caterina Vicentini, Rodrigo Entrialgo-Cadierno, Karmele Valencia, Elisabet Guruceaga, Purvesh Khatri, Vicenzo Corbo, Silvestre Vicent Cambra, Mariano Ponz-Sarvise. Identification of a LAMC2-regulated network featuring targetable effectors for dual therapies in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-089.

Expression dynamics of basement membrane-like extracellular matrix and fibronectin regulate transitions between solid (moruloid) and cavitational (blastuloid) phenotypes of ovarian cancer spheroids., Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid “moruloid” clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into “blastuloid” spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.

The principal humic substances present in soils like Humic Acid (HA) and Fulvic Acid (FA) are plant fertility components many of which basically are aromatic in nature. Assaying these components is a challenge. The present study aims at an enquiry to delineate aromaticity of soil extracted HA and extracted FA by capturing their odor. Since these soil components are bound to soil particles, an effort has been put to find out whether they are more released in dry or wet soil conditions. The aromaticity determination was explored by Electronic nose (E-nose) equipment that had specific array of metal oxide sensors. The other objective of the study was to profile the aromaticity of HA and FA by their extraction from garden soil and its subsequent amendment. The series of experiments comprised of extraction of HA and FA and profiling of Norm Aroma Index (NAI) by E-nose in both wet and dry conditions of soil. The findings were that the yield of HA was more compared to FA; the NAI noted was high in wet condition of the soil compared to dry soil; HA showed more aromaticity compared to FA; the amended soil showed more aromaticity compared to non-amended normal soils. Among the amendment exercises, HA amended soil showed maximum aromaticity. E-nose employment could indicate extent of HA and FA in soils.

In this paper, the performance of iterative turbo coding with nonlinearly distorted orthogonal frequency division multiplexing (OFDM) signal is analyzed. System level implementation in Simulink is performed and bit error rate (BER) performance is analyzed. Initially, we have implemented both, the series turbo coder and iterative turbo coder, compared the performance and then using better performed iterative coder, model is implemented in Simulink. In the model, we introduced highly nonlinear distortion signal using solid state power amplifier (SSPA). It is observed that nonlinear distortion results in very high BER. However, BER performance is significantly improved by the use of iterative turbo coder. It is also seen that 6 iterations is found to be optimum. Higher or smaller iteration results in reduced performance.