Engineering Approaches To Analyze And Target Cancer Invasion And Metastasis

Metastasis of primary tumor accounts for 90% of all deaths in cancer patients. Interaction between cells in the primary tumor is known to play an important role in determining the metastatic potential of cancer cells that leave the primary site. Cancer cells that metastasize through the bloodstream invade through the basement membrane surrounding the primary tumor to enter the adjacent blood capillaries. Then, they can evade the host immune response in the circulation and interact with E-selectin on endothelial cells lining the blood vessel wall to exit the circulation and establish at a secondary site. The first focus of my project was to study the effect of homotypic and heterotypic cell-cell interactions on the metastatic potential of cancer cells using a 3D tumor spheroid model. 3D spheroids generated by culturing breast cancer cell lines on polydimethylsiloxane had stronger interaction with E-selectin, increased invasiveness and resistance to apoptosis inducing signals in the circulation when compared to their respective 2D monolayer grown counterparts on tissue culture plate. Cancer cells entering the lymphatic system get lodged within the tumor draining lymph nodes (TDLN), thus contributing to the lymphatic spread of cancer. Natural killer (NK) cells in the TDLN elicit an anti-tumor response by expressing Tumor necrosis factor-[alpha] Related Apoptosis Inducing Ligand (TRAIL) on their surface. TRAIL can bind to death receptors on cancer cells and induce apoptosis. Despite the presence of immunoregulatory NK cells, lymph node metastases are prevalent in several types of cancers. This is because of the abnormalities of NK cells in the TDLN, including reduced count and decreased cytotoxicity. Given the coexistence of cancer cells and NK cells within the TDLN, the second part of this dissertation was focused on demonstrating a nanomedicine-based approach to enhance the therapeutic efficacy of endogenous NK cells by coating them with liposomes functionalized with TRAIL. Human NK cells coated with TRAIL liposomes were able to induce apoptosis in cancer cells cultured in engineered lymph node microenvironments. Liposomes functionalized with TRAIL and an antibody against mouse NK cells were carried to the TDLN and prevented the lymphatic spread of a subcutaneous tumor in mice

DEVELOPMENT AND APPLICATION OF ELECTRON SPIN RESONANCE (ESR) SPECTROSCOPY TO STUDY INTERACTIONS IN BIOMEMBRANES

Electron Spin Resonance (ESR) spectroscopy has been extensively used to study membrane dynamics and structure. ESR has been shown to be sensitive to a wide range of molecular motions (105 to 1012 s-1). The use of multi-frequency ESR has helped analyze the complex modes of motions typically observed for spin probes attached to biomolecules. Further, the use of pulsed two dimensional ESR techniques (e.g. 2D-ELDOR) has increased the sensitivity to detect molecular motions and has helped to better resolve spectral features. The use of 2D-ELDOR at high frequencies has been particularly challenging due to relatively short dead times required (T2 minimum ~4 ns). We have developed a 95 GHz high power spectrometer which can be operated at room temperatures to study ‘lossy” aqueous samples. We have used this technique to study the partitioning of TEMPO into DMPC. We show that 95 GHz 2D-ELDOR can resolve the spectra of TEMPO in water and the lipid phase. Further, for the first time, we have observed the exchange dynamics of TEMPO between these phases and estimate it to have an exchange rate of 0.5 µs-1. We have then used multi-frequency ESR to study the formation of nanoscopic, co-existing Lo & Ld domains in ternary lipid mixtures. We find that formation of nanodomains does not affect the phase behavior, but indicates the presence of a transition “boundary” region that has intermediate order between the Lo & Ld domains. We further observe that crosslinking of biotinylated gramicidin in these nanodomains, affects the phase behavior by reducing the ordering in the system, but does not change the phase behavior in the macrodomain region. We have also studied the effect of cholesterol depletion in plasma membrane vesicles obtained from RBL cells, and observe that this increases the fraction of the Ld phase in the system. Finally we have used pulsed dipolar ESR to study the crosslinked aggregates of IgE, and have determined that inter IgE-IgE distances to be about 3 - 4 nm. All these results show the ability of ESR to address a wide range of questions related to membrane biophysics

Microenvironment of Tumor-Draining Lymph Nodes: Opportunities for Liposome-Based Targeted Therapy

The World Health Organization (WHO) recently reported that the total number of global cancer cases in 2013 reached 14 million, a 10% rise since 2008, while the total number of cancer deaths reached 8.2 million, a 5.2% increase since 2008. Metastasis is the major cause of death from cancer, accounting for 90% of all cancer related deaths. Tumor-draining lymph nodes (TDLN), the sentinel nodes, are the first organs of metastasis in several types of cancers. The extent of metastasis in the TDLN is often used in disease staging and prognosis evaluation in cancer patients. Here, we describe the microenvironment of the TDLN and review the recent literature on liposome-based therapies directed to immune cells within the TDLN with the intent to target cancer cells

Outcomes of endoscopic third ventriculostomy (ETV) and ventriculoperitoneal shunt (VPS) in the treatment of paediatric hydrocephalus: Systematic review and meta-analysis

Purpose To evaluate the outcomes of endoscopic third ventriculostomy (ETV) and ventriculoperitoneal shunt (VPS) in the treatment of paediatric hydrocephalus. Methods We searched PubMed, MEDLINE, and Cochrane Central Register of Controlled Trials databases for articles published from 2000 to May 2023 (last search date May 6, 2023). Keywords searched included “endoscopic third ventriculostomy”, “ventriculoperitoneal shunting”, “paediatric population”, and “outcomes”. Using random-effects models, we compared success rates and complications of ETV and VPS. The primary outcome was ETV vs.VPS success rates, and the secondary outcome was post-treatment complications. Included studies reported on treatment success and complication rates. Results Out of 126 articles, 8 RCTs and 1 prospective study were included. Six studies reported primary outcome data (806 patients identified: 464 in ETV group, 342 in VPS group). Combined success rates were 81.8% (n = 283/346) for ETV and 86.7% (n = 182/210) for VPS (median follow-up 41 months). There was no difference in success rates between ETV and VPS groups (risk ratio 0.84, 95% confidence interval 0.80–0.90, I2 = 0%, p = 0.93). Combined complication rates were 4.6% (n = 16/346) in the ETV group and 27.1% (n = 57/210) in the VPS group. ETV had a lower rate of postoperative complications (risk ratio 0.76, 95% confidence interval 0.42–1.38, I2 = 53%, p = 0.04). Conclusions Both ETV and VPS are viable surgical options for the management of paediatric hydrocephalus with similar success rates when used as first-line treatment. However, our study concluded that VPS results in a higher complication rate. Registration This systematic review and meta-analysis was formally registered in the PROSPERO International database under the registration number CRD42023452907 on the 29th of August 2023

Resonant Waveguide Grating Imager For Single Cell Monitoring Of The Invasion Of 3D Speheroid Cancer Cells Through Matrigel

The invasion of cancer cells through their surrounding extracellular matrices is the first critical step to metastasis, a devastating event to cancer patients. However, in vitro cancer cell invasion is mostly studied using two-dimensional (2D) models. Three-dimensional (3D) multicellular spheroids may offer an advantageous cell model for cancer research and oncology drug discovery. This chapter describes a label-free, realtime, and single-cell approach to quantify the invasion of 3D spheroid colon cancer cells through Matrigel using a spatially resolved resonant waveguide grating imager

Manufacturing DNA in E. coli yields higher-fidelity DNA than in vitro enzymatic synthesis

Biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceuticals. The quality of starting material plays a pivotal role in determining final product quality. Here, we examined the fidelity of DNA replication using enzymatic methods (in vitro) compared to plasmid DNA produced in vivo in E. coli. Next-generation sequencing approaches rely on in vitro polymerases, which have inherent limitations in sensitivity. To address this challenge, we introduce a novel assay based on loss-of-function (LOF) mutations in the conditionally toxic sacB gene. Our findings show that DNA production in E. coli results in significantly fewer LOF mutations (80- to 3,000-fold less) compared to enzymatic DNA replication methods such as polymerase chain reaction (PCR) and rolling circle amplification (RCA). These results suggest that using DNA produced by PCR or RCA may introduce a substantial number of mutation impurities, potentially affecting the quality and yield of final pharmaceutical products. Our study underscores that DNA synthesized in vitro has a significantly higher mutation rate than DNA produced traditionally in E. coli. Therefore, utilizing in vitro enzymatically produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while using DNA starting material derived from E. coli substantially mitigates this risk

TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids.

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway. We show that inhibition of the COX-2/PGE₂ pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance

Phenotypic switch in blood: effects of pro-inflammatory cytokines on breast cancer cell aggregation and adhesion.

Hematogeneous metastasis can occur via a cascade of circulating tumor cell adhesion events to the endothelial lining of the vasculature, i.e. the metastatic cascade. Interestingly, the pro-inflammatory cytokines IL-6 and TNF-α, which play an important role in potentiating the inflammatory cascade, are significantly elevated in metastatic breast cancer (BCa) patients. Despite their high metastatic potential, human breast carcinoma cells MDA-MB-231 lack interactions with E-selectin functionalized surfaces under physiological shear stresses. We hypothesized that human plasma, 3-D tumor spheroid culture, and cytokine-supplemented culture media could induce a phenotypic switch that allows BCa cells to interact with E-selectin coated surfaces under physiological flow. Flow cytometry, immunofluorescence imaging, and flow-based cell adhesion assay were utilized to investigate the phenotypic changes of MDA-MB-231 cells with various treatments. Our results indicate that plasma, IL-6, and TNF-α promote breast cancer cell growth as aggregates and induce adhesive recruitment of BCa cells on E-selectin coated surfaces under flow. 3-D tumor spheroid culture exhibits the most significant increases in the interactions between BCa and E-selectin coated surfaces by upregulating CD44V4 and sLe(x) expression. Furthermore, we show that IL-6 and TNF-α concentrations in blood may regulate the recruitment of BCa cells to the inflamed endothelium. Finally, we propose a mechanism that could explain the invasiveness of 'triple-negative' breast cancer cell line MDA-MB-231 via a positive feedback loop of IL-6 secretion and maintenance. Taken together, our results suggest that therapeutic approaches targeting cytokine receptors and adhesion molecules on cancer cells may potentially reduce metastatic load and improve current cancer treatments