This document discusses a proposed method to remove tumor-secreted exosomes from the blood to prevent cancer metastasis. Specifically, it aims to:
1. Isolate the protein on Kupffer cells that pancreatic cancer exosomes bind to via integrin proteins.
2. Create antibodies that bind to the epidermal growth factor receptor (EGFR) biomarker found on pancreatic cancer exosomes.
3. Use a modified dialysis machine with microfluidic chips containing the isolated Kupffer cell proteins and EGFR antibodies to filter pancreatic cancer exosomes from patient blood.
This method aims to prevent exosomes from forming pre-metastatic niches and promoting cancer growth and spread by
Objective: Tongue squamous cell carcinoma (TSCC) is a prominent type of oral cancer. Despite the numerous research studies on SCC and microRNAs (miRs), the relation between TSCC and miR-135b-5p is poorly discussed. This experiment aims to find out the possible effect of miR-135b-5p on TSCC with the network of its downstream genes.
Study Design: TSCC tissues and adjacent normal tissues were harvested. Then, expression of miR-135b-5p and AT-rich interactive domain‑containing protein 1A gene (ARID1A) and the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) pathway was analyzed. After the transfection of miR-135b-5p inhibitor and its negative control into TSCC cells, functional assays were employed to measure cell proliferation, apoptosis, and cycle. Next, the target relation between miR-135b-5p and ARID1A was confirmed. In addition, the fact that miR-135b-5p promoted TSCC development via mediating ARID1A was demonstrated by functional rescue experiment.
Results: miR-135b-5p was upregulated in TSCC tissues and cells, while ARID1A was suppressed (p< 0.05). Silenced miR-135b-5p discouraged TSCC cell proliferation, improved apoptosis, induced cell cycle arrest, and increased ARID1A expression while inactivating the PI3K/AKT axis (p<0.05). Furthermore, knockdown of ARID1A reversed the impacts on TSCC cell proliferation and apoptosis exerted by silencing miR-135b-5p.
Conclusion: This research supported that silenced miR-135b-5p impeded TSCC proliferation and apoptosis by promoting ARID1A and inactivating the PI3K/AKT axis, which may provide some indications for TSCC alleviation.
Keywords: apoptosis; ARID1A; ARID1A protein, human; carcinoma, squamous cell; cell line, tumor; cell proliferation; drug resistance, neoplasm; microRNA-135b-5p; microRNAs; PI3K/AKT pathway; neoplasm metastasis; neoplastic stem cells; proliferation; protein binding; tongue; tongue squamous cell carcinoma
Objective: To analyze the sonographic features of different histopathological subtypes of borderline ovarian tumors (BOTs) confirmed by pathology, and to study the ultrasound performances of various types in borderline ovarian tumors.
Study Design: Retrospective analysis was performed on the pathological results and ultrasound projection findings of 129 patients diagnosed as BOTs by ultrasound department of our hospital from January 2012 to November 2019. All patients were confirmed by surgical pathology and scanned consecutively by the investigators using transabdominal or transvaginal ultrasound examination.
Results: Serous borderline tumors (SBOTs) were observed, and the prevalence rate (53%) was significantly higher than that of other subtypes, and the probability of bilateral lesions was higher (40%). The sonogram often showed ultrasound features of papillary neoplasm in the lesion and good internal echo (p<0.05). Mucinous borderline ovarian tumors (MBOTs) were mostly unilateral lesions (86%). The prevalence was second only to SBOTs. Histomorphological examinations were divided into gastrointestinal-type and endocervical-type. Among them, the gastrointestinal type of MBOTs were mostly unilateral, and their incidence was higher than that of endocervical-type of MBOTs. Compared with other pathological subtypes, the gastrointestinal type is more likely to show the sonographic characteristics of huge space occupying in the pelvic and abdominal cavity (mean diameter >10 cm), polycystic, multiple septums, and poor internal echo (p<0.05). The ultrasonographic features of the endocervical-type of MBOTs were similar to those of SBOTs. Compared with gastrointestinal type, the sonographic images showed smaller lesion diameter, less septal or cyst, and more papillary excrescences in the tumor (p<0.05). The borderline clear cell tumor is the intermediate transition between the clear cell adenofibroma and the clear cell carcinoma. The clinical manifestations are diverse and lack specificity. The histology of sonography was mainly solid, and the multiple microcapsules were honeycomb-like. It can also be shown as cystic. Among the 169 patients with BOTs, 20 cases of SBOTs, 17 cases of MBOTs, and 10 cases of other rare subtypes were complicated with other diseases or multiple subtypes. This study did not find significant ultrasonic characteristics were used for distinguish them from other subtypes.
Conclusion: BOTs is a common disease in women during the reproductive period. It is characterized by the development of malignant tumors. Its clinical and pathological subtypes are complex and diverse. It leads many doctors to use the terms “large pelvic mass” and “solid ovarian mass” for diagnosis because of their lack of experience and understanding.
Keywords: adenocarcinoma, mucinous; adenocarcinoma, serous; borderline ovarian tumors; diagnostic imaging; ovarian neoplasms; papillary neoplasms; prognosis; transvaginal ultrasound, ultrasonography
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Objective: Tongue squamous cell carcinoma (TSCC) is a prominent type of oral cancer. Despite the numerous research studies on SCC and microRNAs (miRs), the relation between TSCC and miR-135b-5p is poorly discussed. This experiment aims to find out the possible effect of miR-135b-5p on TSCC with the network of its downstream genes.
Study Design: TSCC tissues and adjacent normal tissues were harvested. Then, expression of miR-135b-5p and AT-rich interactive domain‑containing protein 1A gene (ARID1A) and the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) pathway was analyzed. After the transfection of miR-135b-5p inhibitor and its negative control into TSCC cells, functional assays were employed to measure cell proliferation, apoptosis, and cycle. Next, the target relation between miR-135b-5p and ARID1A was confirmed. In addition, the fact that miR-135b-5p promoted TSCC development via mediating ARID1A was demonstrated by functional rescue experiment.
Results: miR-135b-5p was upregulated in TSCC tissues and cells, while ARID1A was suppressed (p< 0.05). Silenced miR-135b-5p discouraged TSCC cell proliferation, improved apoptosis, induced cell cycle arrest, and increased ARID1A expression while inactivating the PI3K/AKT axis (p<0.05). Furthermore, knockdown of ARID1A reversed the impacts on TSCC cell proliferation and apoptosis exerted by silencing miR-135b-5p.
Conclusion: This research supported that silenced miR-135b-5p impeded TSCC proliferation and apoptosis by promoting ARID1A and inactivating the PI3K/AKT axis, which may provide some indications for TSCC alleviation.
Keywords: apoptosis; ARID1A; ARID1A protein, human; carcinoma, squamous cell; cell line, tumor; cell proliferation; drug resistance, neoplasm; microRNA-135b-5p; microRNAs; PI3K/AKT pathway; neoplasm metastasis; neoplastic stem cells; proliferation; protein binding; tongue; tongue squamous cell carcinoma
Objective: To analyze the sonographic features of different histopathological subtypes of borderline ovarian tumors (BOTs) confirmed by pathology, and to study the ultrasound performances of various types in borderline ovarian tumors.
Study Design: Retrospective analysis was performed on the pathological results and ultrasound projection findings of 129 patients diagnosed as BOTs by ultrasound department of our hospital from January 2012 to November 2019. All patients were confirmed by surgical pathology and scanned consecutively by the investigators using transabdominal or transvaginal ultrasound examination.
Results: Serous borderline tumors (SBOTs) were observed, and the prevalence rate (53%) was significantly higher than that of other subtypes, and the probability of bilateral lesions was higher (40%). The sonogram often showed ultrasound features of papillary neoplasm in the lesion and good internal echo (p<0.05). Mucinous borderline ovarian tumors (MBOTs) were mostly unilateral lesions (86%). The prevalence was second only to SBOTs. Histomorphological examinations were divided into gastrointestinal-type and endocervical-type. Among them, the gastrointestinal type of MBOTs were mostly unilateral, and their incidence was higher than that of endocervical-type of MBOTs. Compared with other pathological subtypes, the gastrointestinal type is more likely to show the sonographic characteristics of huge space occupying in the pelvic and abdominal cavity (mean diameter >10 cm), polycystic, multiple septums, and poor internal echo (p<0.05). The ultrasonographic features of the endocervical-type of MBOTs were similar to those of SBOTs. Compared with gastrointestinal type, the sonographic images showed smaller lesion diameter, less septal or cyst, and more papillary excrescences in the tumor (p<0.05). The borderline clear cell tumor is the intermediate transition between the clear cell adenofibroma and the clear cell carcinoma. The clinical manifestations are diverse and lack specificity. The histology of sonography was mainly solid, and the multiple microcapsules were honeycomb-like. It can also be shown as cystic. Among the 169 patients with BOTs, 20 cases of SBOTs, 17 cases of MBOTs, and 10 cases of other rare subtypes were complicated with other diseases or multiple subtypes. This study did not find significant ultrasonic characteristics were used for distinguish them from other subtypes.
Conclusion: BOTs is a common disease in women during the reproductive period. It is characterized by the development of malignant tumors. Its clinical and pathological subtypes are complex and diverse. It leads many doctors to use the terms “large pelvic mass” and “solid ovarian mass” for diagnosis because of their lack of experience and understanding.
Keywords: adenocarcinoma, mucinous; adenocarcinoma, serous; borderline ovarian tumors; diagnostic imaging; ovarian neoplasms; papillary neoplasms; prognosis; transvaginal ultrasound, ultrasonography
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Objective: To probe into the influence of miR-21 on the proliferation as well as apoptosis of oral squamous cell carcinoma (OSCC) and its causative role.
Study Design: We adopted microarray for detecting the differentially expressed genes in OSCC tumor tis-sues and paracancerous tissues. We assessed the link of miR-21 expression with tumor size, lymph node metastasis, and tumor differentiation. We employed CCK-8 and EdU assay for detecting the impact of miR-21 inhibitor and miR-21 mimic on Cal-27 cell proliferation, as well as TUNEL and AnnexinV-FITC/PI double staining for detecting miR-21 expression on cell apoptosis. We forecasted the possible target of miR-21 via TargetScan, as well as detected the interaction of miR-21 with PTEN via luciferase reporter experiment. The function of miR-21 expression in PTEN signaling pathway was monitored via western blot. We constructed PTEN overexpression plasmid and conducted rescue experiment to evaluate overexpressed PTEN on miR-21–induced proliferation.
Results: Microarray and RT-qPCR indicated that miR-21 expression increased demonstrably in OSCC. Subsequently, statistical analysis showed that miR-21 expression was plainly correlated with tumor size, lymph node metastasis, tumor differentiation, and smoking history. CCK-8 and EdU method exhibited that miR-21 mimics manifestly promoted Cal-27 cell proliferation, while miR-21 inhibitor blatantly inhibited Cal-27 cell proliferation. TUNEL and V-FITC/PI double staining assay showed that miR-21 inhibitor conspicuously promoted Cal-27 cell apoptosis. CCK-8 and EdU assay exhibited that overexpressed PTEN abolished the pro-proliferation influence of miR-21 mimic. TUNEL and V-FITC/PI experiments pointed out that knocking down PTEN abrogated the pro-apoptosis impact of miR-21 inhibitor.
Conclusion: miR-21 contributes to OSCC cell proliferation via targeting PTEN and inhibits its apoptosis.
Keywords: Akt/PKB signaling pathway; apoptosis; biomarkers, tumor; carcinoma, squamous cell; cell line, tumor; cell proliferation; microRNAs; miR-21; miRNA-21; mouth neoplasms; oral cancer; oral squamous cell carcinoma; proliferation; real time PCR
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
Nanodroplet processing platform for deep and quantitative proteome profiling ...Gul Muneer
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
Assessing the clinical utility of cancer genomic and proteomic data across tu...Gul Muneer
Molecular profiling of tumors promises to advance the clinical
management of cancer, but the benefits of integrating
molecular data with traditional clinical variables have not been
systematically studied. Here we retrospectively predict patient
survival using diverse molecular data (somatic copy-number
alteration, DNA methylation and mRNA, microRNA and protein
expression) from 953 samples of four cancer types from The
Cancer Genome Atlas project. We find that incorporating
molecular data with clinical variables yields statistically
significantly improved predictions (FDR < 0.05) for three
cancers but those quantitative gains were limited (2.2–23.9%).
Additional analyses revealed little predictive power across
tumor types except for one case. In clinically relevant genes,
we identified 10,281 somatic alterations across 12 cancer types
in 2,928 of 3,277 patients (89.4%), many of which would
not be revealed in single-tumor analyses. Our study provides
a starting point and resources, including an open-access
model evaluation platform, for building reliable prognostic and
therapeutic strategies that incorporate molecular data
The use of genetic engineering technology in animals has been associated with ethical issues, some of which relate to animal welfare. Discuss examples of genetically engineering animals and evaluate the ethical concerns of genetic engineering.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Objective: To probe into the influence of miR-21 on the proliferation as well as apoptosis of oral squamous cell carcinoma (OSCC) and its causative role.
Study Design: We adopted microarray for detecting the differentially expressed genes in OSCC tumor tis-sues and paracancerous tissues. We assessed the link of miR-21 expression with tumor size, lymph node metastasis, and tumor differentiation. We employed CCK-8 and EdU assay for detecting the impact of miR-21 inhibitor and miR-21 mimic on Cal-27 cell proliferation, as well as TUNEL and AnnexinV-FITC/PI double staining for detecting miR-21 expression on cell apoptosis. We forecasted the possible target of miR-21 via TargetScan, as well as detected the interaction of miR-21 with PTEN via luciferase reporter experiment. The function of miR-21 expression in PTEN signaling pathway was monitored via western blot. We constructed PTEN overexpression plasmid and conducted rescue experiment to evaluate overexpressed PTEN on miR-21–induced proliferation.
Results: Microarray and RT-qPCR indicated that miR-21 expression increased demonstrably in OSCC. Subsequently, statistical analysis showed that miR-21 expression was plainly correlated with tumor size, lymph node metastasis, tumor differentiation, and smoking history. CCK-8 and EdU method exhibited that miR-21 mimics manifestly promoted Cal-27 cell proliferation, while miR-21 inhibitor blatantly inhibited Cal-27 cell proliferation. TUNEL and V-FITC/PI double staining assay showed that miR-21 inhibitor conspicuously promoted Cal-27 cell apoptosis. CCK-8 and EdU assay exhibited that overexpressed PTEN abolished the pro-proliferation influence of miR-21 mimic. TUNEL and V-FITC/PI experiments pointed out that knocking down PTEN abrogated the pro-apoptosis impact of miR-21 inhibitor.
Conclusion: miR-21 contributes to OSCC cell proliferation via targeting PTEN and inhibits its apoptosis.
Keywords: Akt/PKB signaling pathway; apoptosis; biomarkers, tumor; carcinoma, squamous cell; cell line, tumor; cell proliferation; microRNAs; miR-21; miRNA-21; mouth neoplasms; oral cancer; oral squamous cell carcinoma; proliferation; real time PCR
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
Nanodroplet processing platform for deep and quantitative proteome profiling ...Gul Muneer
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
Assessing the clinical utility of cancer genomic and proteomic data across tu...Gul Muneer
Molecular profiling of tumors promises to advance the clinical
management of cancer, but the benefits of integrating
molecular data with traditional clinical variables have not been
systematically studied. Here we retrospectively predict patient
survival using diverse molecular data (somatic copy-number
alteration, DNA methylation and mRNA, microRNA and protein
expression) from 953 samples of four cancer types from The
Cancer Genome Atlas project. We find that incorporating
molecular data with clinical variables yields statistically
significantly improved predictions (FDR < 0.05) for three
cancers but those quantitative gains were limited (2.2–23.9%).
Additional analyses revealed little predictive power across
tumor types except for one case. In clinically relevant genes,
we identified 10,281 somatic alterations across 12 cancer types
in 2,928 of 3,277 patients (89.4%), many of which would
not be revealed in single-tumor analyses. Our study provides
a starting point and resources, including an open-access
model evaluation platform, for building reliable prognostic and
therapeutic strategies that incorporate molecular data
The use of genetic engineering technology in animals has been associated with ethical issues, some of which relate to animal welfare. Discuss examples of genetically engineering animals and evaluate the ethical concerns of genetic engineering.
Crimson Publishers: Improved Version of Cancer Evo-Dev, a Novel Scientific Hy...CrimsonGastroenterology
Chronic but active inflammation, which is activated and maintained by stimulants such as infection and the interactions of stimulants with genetic predisposition, facilitates the occurrence and recurrence of cancers of various histotypes. Chronic inflammation, apparent or unapparent, is indispensible for the development of most malignancies, which has been clarified in hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC). Based on our previous work and the advances of researches on HBV-induced HCC and other inflammation-associated cancers, we presented the framework of a novel cancer theory termed Cancer Evolution and Development (Cancer Evo-Dev) [1-3]. Actually, Cancer Evo-Dev can be applied in cancers of many histotypes.
Suicide gene therapy is based on the delivery of a gene encoding a cytotoxic protein into tumor cells.
For this, there are two possible strategies:
1. Indirect gene therapy using enzyme-activated pro-drug, which allows the conversion of a pro-drug into a lethal drug into cells.
2. Direct gene therapy using a toxin gene, whose expression can change the stability of the cell membrane and reduce the viability of tumor cells, or correct mutated pro-apoptotic genes, generally tumor suppressor genes that in normal condition induce cell suicide.
Cell, Vol. 100, 57–70, January 7, 2000, Copyright 2000 by Cel.docxzebadiahsummers
Cell, Vol. 100, 57–70, January 7, 2000, Copyright 2000 by Cell Press
The Hallmarks of Cancer Review
evolve progressively from normalcy via a series of pre-Douglas Hanahan* and Robert A. Weinberg†
*Department of Biochemistry and Biophysics and malignant states into invasive cancers (Foulds, 1954).
These observations have been rendered more con-Hormone Research Institute
University of California at San Francisco crete by a large body of work indicating that the ge-
nomes of tumor cells are invariably altered at multipleSan Francisco, California 94143
†Whitehead Institute for Biomedical Research and sites, having suffered disruption through lesions as sub-
tle as point mutations and as obvious as changes inDepartment of Biology
Massachusetts Institute of Technology chromosome complement (e.g., Kinzler and Vogelstein,
1996). Transformation of cultured cells is itself aCambridge, Massachusetts 02142
multistep process: rodent cells require at least two intro-
duced genetic changes before they acquire tumorigenic
competence, while their human counterparts are moreAfter a quarter century of rapid advances, cancer re-
difficult to transform (Hahn et al., 1999). Transgenicsearch has generated a rich and complex body of knowl-
models of tumorigenesis have repeatedly supported theedge, revealing cancer to be a disease involving dy-
conclusion that tumorigenesis in mice involves multiplenamic changes in the genome. The foundation has been
rate-limiting steps (Bergers et al., 1998; see Oncogene,set in the discovery of mutations that produce onco-
1999, R. DePinho and T. E. Jacks, volume 18[38], pp.genes with dominant gain of function and tumor sup-
5248–5362). Taken together, observations of humanpressor genes with recessive loss of function; both
cancers and animal models argue that tumor develop-classes of cancer genes have been identified through
ment proceeds via a process formally analogous to Dar-their alteration in human and animal cancer cells and
winian evolution, in which a succession of geneticby their elicitation of cancer phenotypes in experimental
changes, each conferring one or another type of growthmodels (Bishop and Weinberg, 1996).
advantage, leads to the progressive conversion of nor-Some would argue that the search for the origin and
mal human cells into cancer cells (Foulds, 1954; Nowell,treatment of this disease will continue over the next
1976).quarter century in much the same manner as it has in
the recent past, by adding further layers of complexity
to a scientific literature that is already complex almost An Enumeration of the Traits
beyond measure. But we anticipate otherwise: those The barriers to development of cancer are embodied
researching the cancer problem will be practicing a dra- in a teleology: cancer cells have defects in regulatory
matically different type of science than we have experi- circuits that govern normal cell proliferation and homeo-
enced over the past 25 years. Surely much of this change stasis. T.
Cell, Vol. 100, 57–70, January 7, 2000, Copyright 2000 by Cel.docxketurahhazelhurst
Cell, Vol. 100, 57–70, January 7, 2000, Copyright 2000 by Cell Press
The Hallmarks of Cancer Review
evolve progressively from normalcy via a series of pre-Douglas Hanahan* and Robert A. Weinberg†
*Department of Biochemistry and Biophysics and malignant states into invasive cancers (Foulds, 1954).
These observations have been rendered more con-Hormone Research Institute
University of California at San Francisco crete by a large body of work indicating that the ge-
nomes of tumor cells are invariably altered at multipleSan Francisco, California 94143
†Whitehead Institute for Biomedical Research and sites, having suffered disruption through lesions as sub-
tle as point mutations and as obvious as changes inDepartment of Biology
Massachusetts Institute of Technology chromosome complement (e.g., Kinzler and Vogelstein,
1996). Transformation of cultured cells is itself aCambridge, Massachusetts 02142
multistep process: rodent cells require at least two intro-
duced genetic changes before they acquire tumorigenic
competence, while their human counterparts are moreAfter a quarter century of rapid advances, cancer re-
difficult to transform (Hahn et al., 1999). Transgenicsearch has generated a rich and complex body of knowl-
models of tumorigenesis have repeatedly supported theedge, revealing cancer to be a disease involving dy-
conclusion that tumorigenesis in mice involves multiplenamic changes in the genome. The foundation has been
rate-limiting steps (Bergers et al., 1998; see Oncogene,set in the discovery of mutations that produce onco-
1999, R. DePinho and T. E. Jacks, volume 18[38], pp.genes with dominant gain of function and tumor sup-
5248–5362). Taken together, observations of humanpressor genes with recessive loss of function; both
cancers and animal models argue that tumor develop-classes of cancer genes have been identified through
ment proceeds via a process formally analogous to Dar-their alteration in human and animal cancer cells and
winian evolution, in which a succession of geneticby their elicitation of cancer phenotypes in experimental
changes, each conferring one or another type of growthmodels (Bishop and Weinberg, 1996).
advantage, leads to the progressive conversion of nor-Some would argue that the search for the origin and
mal human cells into cancer cells (Foulds, 1954; Nowell,treatment of this disease will continue over the next
1976).quarter century in much the same manner as it has in
the recent past, by adding further layers of complexity
to a scientific literature that is already complex almost An Enumeration of the Traits
beyond measure. But we anticipate otherwise: those The barriers to development of cancer are embodied
researching the cancer problem will be practicing a dra- in a teleology: cancer cells have defects in regulatory
matically different type of science than we have experi- circuits that govern normal cell proliferation and homeo-
enced over the past 25 years. Surely much of this change stasis. T ...
Discuss an example of knockout mouse model used for disease modelling (Metast...SaniikaRenganadan
Title: Discuss an example of knockout mouse model used for disease modelling
Disease: Metastatic Bladder Cancer
Module: Gene and Tissue Culture Technology
Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovari...ijtsrd
Background and objective: Treatment of epithelial ovarian cancer (EOC) is a major challenge with only 30% 5-year survival rate. The outcome of the different therapeutic modalities is still poor, and there is an urgency to find new treatment lines. The effect of mesenchymal stem cells on different tumors is greatly variable. The present work shows the effect of cord blood mesenchymal stem cells conditioned media (MSC CM) in different concentrations on epithelial ovarian cancer stem cells (CD44+ cells) in vitro. Methodology: Ovarian cancer stem cells were subjected to MSC CM of (100%, 75%, 50%, 25%) concentrations for 72 hours followed by investigation of cell morphology, proliferation, apoptosis, cell cycle and expression of certain genes (Oct-4, Sox-2, and Nanog). Results: Cell shrinkage and cell debris was observed with all cancer cell lines by contrast with control. MTT assay showed a reduction in proliferation, in a concentration-dependent manner. The annexin-v results demonstrated a significant early and late apoptosis. There was an increase in the sub-G1 phase of the cell cycles indicating apoptosis. There was a progressive suppression of embryonic stemness genes in all cell lines compared to control. Conclusion: Based on these results, it was concluded that MSC CM may be a potential ovarian cancer inhibitor that may create a new modalities of treatment in ovarian cancer patients. Maher E. Elgaly | Mohamed E. El Ghareeb | Mohamed H. Bedairy | Ahmed M. Badawy | Abeer Shaaban | Farha El shennawy"Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovarian Cancer Cells in Vitro" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-5 , August 2018, URL: http://www.ijtsrd.com/papers/ijtsrd18182.pdf http://www.ijtsrd.com/other-scientific-research-area/other/18182/cord-blood-mesenchymal-stem-cells-conditioned-media-suppress-epithelial-ovarian-cancer-cells-in-vitro/maher-e-elgaly
Vorinostat combined with DNMTi epigenetically controls the proliferation of l...
biochem of cancer modified dialysis treatment
1. Thomas Brinkman
Modified Dialysis Filtration of Pancreatic Ductal Adenocarcinoma Exosomes to Prevent
Formation of Pre-Metastatic Niche
I. SPECIFIC AIMS
The objective of this study is to devise a method to remove tumor secreted exosomes
from the blood. We have chosen to extract these exosomes from the blood by binding to the
proteins found on Kupffer cells in vitro along with the use of primary antibodies specific to
PDAC exosome biomarker epidermal growth factor receptor (EGFR). Tumorigenic exosomes are
microvesicles secreted by the primary tumor and play roles in tumor development and metastasis.
They do this by promoting carcinogenesis and modifying the immune system to allow the tumor
to circulate in the blood unscathed (An et al., 2015). These microvesicles arrive at metastatic sites
by organ-specific integrins prior to tumor cell arrival. This forms a premetastatic niche for the
tumor in order to decrease exposure time in blood and increase the chances of a successful
metastasis (Offord, 2016). Although exosomes are just beginning to be fully explored, it is known
that in pancreatic ductal adenocarcinoma (PDAC) exosomes bind to proteins on Kupffer cells in
the liver. This binding triggers a cascade of events that promote favorable conditions for
metastasis (Costa-Silva, et al., 2015). EGFR is a biomarker that is commonly overexpressed in
pancreatic cancers. EGFR has been associated with many factors involved in carcinogenesis
(Adamczyk et al., 2011). The aspects of the exosome mediated metastatic pathway that can be a
target in this treatment involves the protein on Kupffer cells that bind with integrin αvβ5 along
with the common biomarker of exosomes, EGFR.
We hypothesize that PDAC exosomes can be successfully removed from the blood using
a modified dialysis treatment. We have chosen to study this pathway in metastatic PDAC cells as
a model system. Previous research suggests that PDAC derived exosomes bind to a protein on
Kupffer cells and trigger a favorable environment for metastasis. In addition, previous literature
suggests that this metastatic cascade can be reduced by the inhibition of exosomal macrophage
migratory inhibitory factor (MIF). This is a protein found in high numbers in the PDAC
exosomes (Offord, 2016), so removal of exosomes would have a similar effect. Data suggests that
PDAC derived exosomes indirectly allow metastasis by interacting with Kupffer cells through
proteins carried within the exosomes. Therefore,the specific aims for this proposal are:
1) Identify and isolate the specific protein on Kupffer cells responsible for PDAC
exosome integrin binding.
2) Create primary antibodiesthat adhere to PDAC exosome biomarker EGFR.
3) Use a modified dialysis machine to pass the patient’s blood through a microfluidic
chip to isolate circulating PDAC-exosomes fromthe blood.
Pancreatic cancer has the lowest five-year survival rate amongst solid tumors, and our
proposal works to prevent the progression of this disease. Data obtained from previous studies
support our hypothesis that exosomes will bind to the isolated receptor proteins from Kupffer
cells and primary antibodies on a microchip. Removing these exosomes from the blood is critical
for an increased survival rate for pancreatic cancer patients. Developing this treatment can allow
for a decrease of metastatic rates and lead to future exosome isolation treatments for other tumor
types using a modified dialysis treatment.
2. II. Introduction
Cancer is a highly studied yet poorly understood disease that requires continual advances
in technology to keep up with its plasticity and survivability. It is projected that in 2016 there will
be 1,685,210 new cases of cancer diagnosed and 595,690 deaths from cancer in the United States
alone (Siegel, et al., 2016). One of the key aspects to tumor survival is its ability to become
dormant and re-metastasize after some time. Dormant tumors are asymptomatic and currently
undetectable through traditional treatments. Dormancy can occur at any period of tumor
development, and may contribute to metastasis and relapse either at the site of the primary tumor
or at distant organs reportedly decades after treatment (Wang & Lin, 2013). The tumors present
upon relapse are thought to be even more malicious than the primary tumor because these cells
survived treatments and have acquired traits favorable to their survival. Pancreatic cancer, as the
main focus of this proposal, is one of the most destructive tumor types with an average survival
rate of about 6 months and a 6% five-year survival rate. (Costa-Silva, et al., 2015). Pancreatic
ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer accounting for
nearly 90% of deaths in pancreatic cancer patients (Costa-Silva, et al., 2015). It is estimated that
53,070 people will be diagnosed and 41,780 will die of pancreatic cancer in the United States in
2016 (American Cancer Society, 2016). With the survival rate almost equal to the death rate,
further study is required to reduce the invasiveness of pancreatic cancer. Fortunately, researchers
have been able to identify a mechanism for metastasis in PDAC cells that may respond to
therapeutic treatments.
Kupffer cells (KC) bind to integrins found on PDAC exosomes, and stimulate the
formation of the premetastatic niche. KC are found in the lumen of liver sinusoids and are known
to activate macrophages (Bilzer, Roggel, & Gerbes, 2006). They are a critical aspect of the host’s
innate immunity. Unfortunately they also induce favorable metastatic environments through the
production of hepatocyte growth factor known to aid in tumor proliferation, and increase
angiogenesis by secreting proteases that change the extracellular matrix (Bilzer, Roggel, &
Gerbes, 2006). It has been found that these events occur upon the binding with integrin αvβ5 on
PDAC exosomes (Offord, 2016). Filtration of integrin αvβ5 may decrease the pro-metastatic
factors secreted by Kupffer cells and lead to a decrease in metastasis for PDAC patients.
Metastasis is defined as migration of tumor cells from the primary tumor to another
location in the body. It is thought that tumors do this in an organ-specific manner. This organ
specificity is known as metastatic organotropism and was first predicted by Stephen Paget in
1889 (Hoshino, et al., 2015). Tumors secrete molecules to aid in their metastasis, and exosomes
may be the pivotal molecule in organ specificity. Tumor secreted exosomes are thought to play a
key role in intercellular communication of cancer cells with future metastatic sites through
signaling molecules, DNA transfer, and microRNA secretion (Costa-Silva, et al., 2015).
Migratory inhibitory factor (MIF) is one of these proteins found in high numbers in PDAC
isolated exosomes. It has been noted that the knockdown of MIF expression decreases the
tumor’s ability to metastasize, suggesting MIF plays a role in exosome-mediated metastasis
(Costa-Silva, et al., 2015). It was found that exosomal integrins direct this metastatic
organotropism by binding to target cells in a tissue-specific manner (Hosino, et al., 2015). It is
known that integrin αvβ5 has a specific binding site in the liver on Kupffer cells (Offord, 2016).
This integrin is known as an upstream regulator of transforming growth factor β (TGFβ). TGFβ is
commonly overexpressed in PDAC patients and known to promote epithelial-to-mesenchymal
transition, angiogenesis, and suppress the immune system’s ability to detect tumors (Hezel, el al.,
2015). TGFβ also promotes the formation of fibronectin by hepatic stellate cells. Fibronectin is a
cell adhesion molecule that creates favorable environment for tumor arrival (Costa-Silva, et al.,
2015). If PDAC exosomes can be removed altogether it would be harder for the tumor to create a
premetastatic niche by removing these metastatic factors. Figure 1 depicts the cascade of events
caused by PDAC exosomes.
3. A PDAC exosomal biomarker called epidermal growth factor receptor (EGFR) is found
overexpressed in pancreatic cancer. It has known effects for carcinogenesis, increased tumor
aggressiveness, and potentially a trigger for pathways involving cell proliferation, differentiation,
and migration (Adamczyk, et al., 2011). This is highly characterized and primary antibodies can
be created for this protein. Primary antibodies can be synthesized by injecting an antigen of
interest into a host animal and its immune system makes an antibody against that specific protein.
A portion of the host animal’s spleen is removed and fused with a myeloma cell. This creates a
hybridoma that will produce continually antibodies for future experimental purposes. A
monoclonal antibody only binds to one epitope on an antigen target, which creates high
specificity for only one type of protein.
Exosome removal techniques are an active area of research and various methods have
been developed. A table of these methods can be seen in Table 1 below. A microfluidic apparatus
has been developed that extracts microvesicles from blood. This was determined to be faster and
cheaper than traditional centrifugation and filtration techniques. It only requires small quantities
of microvesicles in the fluid for successful isolation. The microfluidic technique extracts
exosomes in an antigen-specific manner using a microchip containing antibodies. It was
determined that the efficiency of isolating microvesicles was 42-94% in this microfluidic
approach (Chen, et al., 2010). This was done on a small scale, but this idea can be expanded into
a body-wide treatment through the use of a modified dialysis machine. Dialysis involves
circulating a patient’s blood into a machine. The blood is then passed through a filter and waste
products are cleared to return back to the patient. This can be modified in many ways by using
different combinations of biomarkers commonly found in PDAC exosomes for successful
filtration. This treatment is most beneficial for patients diagnosed with PDAC early on to prevent
PDAC metastasis, and for patients whose tumors have went into remission to prevent future
metastasis. Upon success of this treatment, it can also be expanded to other tumor types by using
their common biomarkers. Success of this apparatus may save millions from potential metastasis
or relapse of cancer.
Source: Offord, 2016
4. Figure 1: Mechanism of PDAC exosomes on Kupffer cells in the liver. The Kupffer cells are
attached to the liver and bind to PDAC exosomes via integrin specific binding. Kupffer cells are
macrophages and upon interaction with PDAC exosomes emits Transforming Growth Factor β
(TGFβ). At this point, the liver’s stellate cells begin secreting fibronectin that recruits bone
marrow derived macrophages. Additional secretions include epithelial cells and fibroblasts. All of
these secretions are important to a premetastatic niche formation favorable for the tumor’s arrival
during metastasis (Offord, 2016).
Table 1: Analysis of exosome removal techniques. Techniques have been performed in various
experiments. Affinity isolation technique appears to be the best. This method has very high
specificity and purity, is relatively cheap, and contamination is not a concern. These reasons
together form the basis of our experimental procedure. (An et al., 2015).
Figure 2:Microchip used for filtration of exosomes. This image depicts the experimental setup
of one microchip used to filter exosomes from the blood. In our experiment there will be 10
microchips hooked up side-by-side creating a parallel circuit for the blood to travel though. See
methods part 3 for further description.
III. Experimental Design
1) Identify and isolate the specific protein on Kupffer cells responsible for PDAC
exosome integrin binding.
i. Experimental procedure: A tissue culture containing a primary cell line of Kupffer
cells will be cultured and maintained on DMEM. A binding assay will be conducted to
determine which receptor binds the αVβ5 integrin found on PDAC exosomes.
Radioactive PDAC exosomes will be placed into tissue cultures and binding will be
Source: An et al, 2015
Source: Chen et al., 2010
5. detected through autoradiography. Nonspecific binding is calculated by doing the
binding assay in the presence of an excess of unlabeled PDAC exosomes. Protocol
modified from Lodish et al.
Once the receptor is determined the Thermo Scientific Cell Surface Protein
Isolation Kit will be used to remove these receptors. Once these receptors are removed
a western blot will be performed to determine if the correct protein has been isolated.
The control group will apply same antibodies used to bind Kupffer cell integrin
receptor onto a different type of tissue such as epithelial cells. This will assure that
there will be no nonspecific binding by these antibodies. A control protein for the
Western Blot will be GAPDH due to its constitutive expression. It will act as a loading
control and verification of the successfulprotein isolation.
ii. Data Analysis: The first part of the data analysis is determining which receptor the
PDAC exosomes are binding to and ultimately the expected number of exosomes each
Kupffer cell can bind to. Calculating the specific and nonspecific binding present will
quantify this. Total binding will be determined by the amount of radiation given off
upon the addition of a known amount of radiolabeled exosome. Nonspecific binding is
calculated by the amount of labeled PDAC exosomes bound in the presence of pre-
added unlabeled PDAC exosomes. Since there are only a certain number of receptors
per cell, they will get saturated by the unlabeled PDAC exosomes. Then with addition
of labeled exosome, the nonspecific binding sights will be labeled since they will not
be saturated. Specific binding will be determined by finding the difference between
total binding and nonspecific binding. With this data a curve can be generated.
Saturation values will be determine will allow the determination of the amount of
PDAC exosome present that these receptors can successfully bind to.
The Western Blot analysis will be analyzed after isolation of the receptor cells.
Band presence in experimental group will be compared to control for presence of
isolated receptor. GAPDH will be used as a loading control. The proteins are known
since the antibody used for the blot is specific to a certain protein.
iii. Expected Results: We expect to see a high presence of specific binding when using
the radioactive exosomes. A positive result would also include a very low amount of
nonspecific binding. We expect a curve that levels off showing saturable binding of
the Kupffer cells with the radiolabeled exosomes. The Western Blot is expected to
detect the presence of a band in the experimental, but the absence of this band in the
control section. When the antibodies are used on epithelial cells it is expected that
there will be no binding because there will be no Kupffer cells present. Constitutive
expression is expected in the GAPDH section to assure the loading was equal and the
cell is still alive and metabolizing.
iv. Anticipated Complications: This experiment may show problems in the specificity
of the radioactive exosomes used to identify the receptors. If there is low specificity
for the PDAC exosomes, a blocking solution will be applied prior to the addition of
the radiolabeled exosomes. This will allow most proteins in the cell to be saturated
and decrease nonspecific binding. Another possible complication is the structural
integrity of receptors for the PDAC integrin on the surface of Kupffer cells. If they do
not maintain their structure in vitro, then this experiment would have to be done in
vivo.
6. 2) Create primary antibodies that adhere to PDACexosome biomarker EGFR.
i. Experimental Procedure: Epidermal Growth Factor Receptor (EGFR) will be
purchased from Abcam (ab89746). Rabbits will first be immunized with the EGFR
protein and left for three weeks to allow antibodies to form. A booster shot will be
administered 3 weeks after the initial immunization and then 3 additional days will
pass before fusion. After this, the spleen will be removed from the animals. A
screening assay will be performed to assure the correct antibodies are produced from
the B cells. The screening assay and the assay for making a hybridoma were
modified from Lerner, 1981. Cells from the spleen will be fused with a human
myeloma cell because most spleen cells will be making the antibody of interest.
Spleen and myeloma cells will be fused using the fusing agent polyethylene glycol.
This creates the hybridoma that will secrete the antibody of interest. Once the
hybridoma colonies are formed and isolated, the cells are then screened again to
ensure the production of the antibodies. Antibodies will be tested for quality by
staining tissue with EGFR. Control will consist of mixing antibodies with EGFR
antigen in a tube then staining tissue with EGFR through immunohistochemistry to
test specificity of antibodies. Another control will mix the antibody made with
antigens that are similar to EGFR and staining tissue-containing EGFR again.
ii. Data Analysis: During the screening assay to assure the correct antibodies are
produced, a gamma counter will be used. Background counts will be compared to
the positive antibody containing wells to verify the presence of antibodies. The
presence of staining will be searched for when observing the tests of the quality of
the antibody and specificity.
iii. Expected Results: The gamma counter should read around 100 cpm for the
background control. Then with the positive wells the gamma read should be
anywhere from 200 to 2,000 cpm. This should be the result for both screens
performed to ensure a successful hybridoma has been created. This would indicate
the presence of our antibody against the EGFR, and allow for further analysis. There
should be staining present in the experimental antibody staining. Then for the
control when the antibody is mixed with the EGFR antigen, it should not stain any
tissue to indicate these antibodies created are specific to EGFR. The other control
with similar antignes should have staining found on the tissue with EGFR to
indicate the antibody for EGFR does not bind to any similar antigens.
iv. Anticipated Complications: The creation of the hybridoma is based off of the
spleen cells creating the antibody in question. If the host’s immune response is weak
it will result in little to no antibody production. In this case, the experiment would
need to be restarted using a new host. This is why screening is crucial for the
experiment to proceed. In creating the hybridoma, the perfect myeloma cell line
must be chosen. The human myeloma should work, but it is not certain that this will
fuse and create the hybridoma. If the spleen cells and the myeloma do not fuse then
a more careful selection must be made for which myeloma will be used for
production of the hybridoma. If specificity is a problem in these experiments, a new
antibody will have be created for a different biomarker on PDAC exosomes.
3) Use a modified dialysis machine to pass the patient’s blood through a
microfluidic chip to isolate circulating PDAC-exosomes from the blood.
7. i. Experimental Procedure: Microfluidic chips that were created by Chen et al. found
in references below will be used for filtration of PDAC exosomes. A sample of how
one microchip assay looks can be found in Figure 3 above. The difference in our
chip is the presence of the receptors on Kupffer cells that bind integrin αvβ5 of
PDAC exosomes along with the antibody created for EGFR. The control group will
have chips containing anti-EGFR and anti- Kupffer integrin receptor antibodies as
negative controls. An additional control will be non-cancerous mouse blood running
through experimental chips. These chips have an average flow rate of 16 uL/min, so
using ten chips in a parallel circuit would cause a flow rate of 160 uL/min. The initial
tests would be on mice with known PDAC cancer for experimental purposes. A
mouse has an average blood volume of 1.5 mL, so it would take about 10 minutes for
one round of blood to circulate through the machine. The protocol was modified from
Chen et al., 2010. This will go on for 2 hours to ensure blood has circulated through
microchips multiple times. Standard dialysis cannulation protocol would be followed
to get blood circulating through the machine and back into the patient. The dialysis
machine is used to extract the blood and create the external circuit for blood to go
through the chips. When the treatment has concluded, electron micrograph (EM)
images will be taken to visualize exosomes. Then DNA and RNA will be isolated
from the microvessicles in the chips. Subsequent PCR and RT-qPCR will be
performed using primers of known genes found in PDAC exosomes. DNA marker
tested will be KRAS, and the miRNA genes will be miR-17-5p and miR-21 (An, et
al. 2015). These are biomarkers found in pancreatic cancer microvessicles. Products
of PCR will be run on gel electrophoresis for analysis.
ii. Data Analysis: EM images will allow for the visualization of the presence of
exosomes and determination of the size specs of the exosomes isolated. Microchips
will be analyzed for the treatment and control groups. Gel electrophoresis will
separate DNA based on molecular size to indicate the presence of our genes of
interest.
iii. Expected Results: EM analysis should show the appearance of tiny beads
(exosomes) throughout the experimental chips. The control chips containing anti-
EGFR and anti-Kupffer Cells should not contain exosomes. Size of these exosomes
will be determined from these images. For the PCR gel electrophoresis results there
should be bands found where the DNA and miRNA is specific to PDAC exosomes.
Bands should not be found in the noncancerous blood dialysis treatment to confirm
specificity of the isolation. Since the current of blood was divided equally, the
number of exosomes and quantity of isolated DNA/miRNA per chip should be equal
per chip.
iv. Anticipated Complications: If the EM images show little to no PDAC exosomes
isolated, the time period the treatment is run will be adjusted. The primary concern
with prolonged dialysis running is that blood current may sweep some exosomes off
of the chip and bring it back into the blood. If this is the case and shorter running
times do resolve this, then more specific antibodies and binding proteins will need to
be designed for higher specificity. If the normal blood treatment control shows
isolated exosomes, then nonspecific binding is occurring. Proteins used in the assay
to bind the exosomes would have to be changed. If bands from the PCR of the
miRNA biomarkers do not appear, then the DNA will be the only isolation performed
because miRNA is unstable and easily degraded with RNases found ubiquitously. If
8. the amount of DNA/miRNA isolated from the chips is not near equal, a modification
in the number or quality of chips will be changed to enhance results.
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