Person:

Sengupta, Shiladitya

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 or IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1(\alpha) and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies. Understanding the mechanisms that are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.

Despite the advances in the design of antibody–drug conjugates (ADCs), the search is still ongoing for novel approaches that lead to increased stability and homogeneity of the ADCs. We report, for the first time, an ADC platform technology using a platinum(ii)-based linker that can re-bridge the inter-chain cysteines in the antibody, post-reduction. The strong platinum–sulfur interaction improves the stability of the ADC when compared with a standard maleimide-linked ADC thereby reducing the linker–drug exchange with albumin significantly. Moreover, due to the precise conserved locations of cysteines, both homogeneity and site-specificity are simultaneously achieved. Additionally, we demonstrate that our ADCs exhibit increased anticancer efficacy in vitro and in vivo. The Pt-based ADCs can emerge as a simple and exciting proposition to address the limitations of the current ADC linker technologies.

Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Background: Pancreatic cancer remains the deadliest of all cancers, with a mortality rate of 91%. Gemcitabine is considered the gold chemotherapeutic standard, but only marginally improves life-span due to its chemical instability and low cell penetrance. A new paradigm to improve Gemcitabine’s therapeutic index is to administer it in nanoparticles, which favour its delivery to cells when under 500 nm in diameter. Although promising, this approach still suffers from major limitations, as the choice of nanovector used as well as its effects on Gemcitabine intracellular trafficking inside pancreatic cancer cells remain unknown. A proper elucidation of these mechanisms would allow for the elaboration of better strategies to engineer more potent Gemcitabine nanotherapeutics against pancreatic cancer. Methods: Gemcitabine was encapsulated in two types of commonly used nanovectors, namely poly(lactic-co-glycolic acid) (PLGA) and cholesterol-based liposomes, and their physico-chemical parameters assessed in vitro. Their mechanisms of action in human pancreatic cells were compared with those of the free drug, and with each others, using cytotoxity, apoptosis and ultrastructural analyses. Results: Physico-chemical analyses of both drugs showed high loading efficiencies and sizes of less than 200 nm, as assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), with a drug release profile of at least one week. These profiles translated to significant cytotoxicity and apoptosis, as well as distinct intracellular trafficking mechanisms, which were most pronounced in the case of PLGem showing significant mitochondrial, cytosolic and endoplasmic reticulum stresses. Conclusions: Our study demonstrates how the choice of nanovector affects the mechanisms of drug action and is a crucial determinant of Gemcitabine intracellular trafficking and potency in pancreatic cancer settings.

Cancer microenvironment is a remarkably heterogeneous composition of cellular and non-cellular components, regulated by both external and intrinsic physical and chemical stimuli. Physical alterations driven by increased proliferation of neoplastic cells and angiogenesis in the cancer microenvironment result in the exposure of the cancer cells to elevated levels of flow-based shear stress. We developed a dynamic microfluidic cell culture platform utilizing eshopagael cancer cells as model cells to investigate the phenotypic changes of cancer cells upon exposure to fluid shear stress. We report the epithelial to hybrid epithelial/mesenchymal transition as a result of decreasing E-Cadherin and increasing N-Cadherin and vimentin expressions, higher clonogenicity and ALDH positive expression of cancer cells cultured in a dynamic microfluidic chip under laminar flow compared to the static culture condition. We also sought regulation of chemotherapeutics in cancer microenvironment towards phenotypic control of cancer cells. Such in vitro microfluidic system could potentially be used to monitor how the interstitial fluid dynamics affect cancer microenvironment and plasticity on a simple, highly controllable and inexpensive bioengineered platform.

Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. Here we report the engineering of personalized tumour ecosystems that contextually conserve the tumour heterogeneity, and phenocopy the tumour microenvironment using tumour explants maintained in defined tumour grade-matched matrix support and autologous patient serum. The functional response of tumour ecosystems, engineered from 109 patients, to anticancer drugs, together with the corresponding clinical outcomes, is used to train a machine learning algorithm; the learned model is then applied to predict the clinical response in an independent validation group of 55 patients, where we achieve 100% sensitivity in predictions while keeping specificity in a desired high range. The tumour ecosystem and algorithm, together termed the CANScript technology, can emerge as a powerful platform for enabling personalized medicine.

Understanding the emerging models of adaptive resistance is key to overcoming cancer chemotherapy failure. Using human breast cancer explants, in vitro cell lines, mouse in vivo studies and mathematical modelling, here we show that exposure to a taxane induces phenotypic cell state transition towards a favoured transient CD44HiCD24Hi chemotherapy-tolerant state. This state is associated with a clustering of CD44 and CD24 in membrane lipid rafts, leading to the activation of Src Family Kinase (SFK)/hemopoietic cell kinase (Hck) and suppression of apoptosis. The use of pharmacological inhibitors of SFK/Hck in combination with taxanes in a temporally constrained manner, where the kinase inhibitor is administered post taxane treatment, but not when co-administered, markedly sensitizes the chemotolerant cells to the chemotherapy. This approach of harnessing chemotherapy-induced phenotypic cell state transition for improving antitumour outcome could emerge as a translational strategy for the management of cancer.

Background: Dandruff is a common scalp condition characterized by excessive scaling and itch. Aberrant colonization of the scalp by commensal Malassezia spp. is a major contributor in the multifactorial etiology of dandruff. Literature based understanding of Malassezia linked pathophysiology of dandruff allowed us to comprehend a strategy to potentiate the efficacy of a known antifungal agent used in dandruff therapy. The aim of this study was to determine the efficacy and skin safety of VB-001 antidandruff leave-on formulation in comparison with marketed antidandruff ZPTO shampoo in patients with moderate adherent dandruff of the scalp. Methods: Healthy males or females aged ≥ 15 years and ≤ 65 with a clinical diagnosis of moderate adherent dandruff of the scalp were recruited for the study to monitor the effects of topical VB-001 versus those of marketed antidandruff ZPTO shampoo. Results: 168 subjects were randomized to the treatment (VB-001, n = 84) and control (ZPTO shampoo, n = 84) groups. The efficacy of each product was evaluated by comparing proportion of subjects who have shown reduction in flaking by ASFS (adherent scalp flaking score) and pruritus by IGA (investigator global assessment) score. VB-001 imparted consistently better reduction in ASFS and enabled early reduction of pruritus in comparison to marketed ZPTO shampoo. Conclusion: VB-001, a leave-on formulation with ingredients chosen to selectively disturb the Malassezia niche on dandruff scalp by denying extra nutritional benefits to the microbe, provides unique advantages over existing best in class ZPTO shampoo therapy. It has the potential to emerge as an attractive novel treatment for moderate adherent dandruff. Trial registration CTRI Registration number: CTRI/2013/01/003283. Registered on: 02/01/2013

Metastasis can involve repeated cycles of epithelial-to-mesenchymal transition (EMT) and its reverse mesenchymal-to-epithelial transition. Cells can also undergo partial transitions to attain a hybrid epithelial/mesenchymal (E/M) phenotype that allows the migration of adhering cells to form a cluster of circulating tumour cells. These clusters can be apoptosis-resistant and possess an increased metastatic propensity as compared to the cells that undergo a complete EMT (mesenchymal cells). Hence, identifying the key players that can regulate the formation and maintenance of such clusters may inform anti-metastasis strategies. Here, we devise a mechanism-based theoretical model that links cell–cell communication via Notch-Delta-Jagged signalling with the regulation of EMT. We demonstrate that while both Notch-Delta and Notch-Jagged signalling can induce EMT in a population of cells, only Jagged-dominated Notch signalling, but not Delta-dominated signalling, can lead to the formation of clusters containing hybrid E/M cells. Our results offer possible mechanistic insights into the role of Jagged in tumour progression, and offer a framework to investigate the effects of other microenvironmental signals during metastasis.

Pharmacological concentrations of small molecule natural products, such as ascorbic acid, have exhibited distinct cell killing outcomes between cancer and normal cells whereby cancer cells undergo apoptosis or necrosis while normal cells are not adversely affected. Here, we develop a mathematical model for ascorbic acid that can be utilized as a tool to understand the dynamics of reactive oxygen species (ROS) induced cell death. We determine that not only do endogenous antioxidants such as catalase contribute to ROS-induced cell death, but also cell membrane properties play a critical role in the efficacy of ROS as a cytotoxic mechanism against cancer cells vs. normal cells. Using in vitro assays with breast cancer cells, we have confirmed that cell membrane properties are essential for ROS, in the form of hydrogen peroxide (H2O2), to induce cell death. Interestingly, we did not observe any correlation between intracellular H2O2 and cell survival, suggesting that cell death by H2O2 is triggered by interaction with the cell membrane and not necessarily due to intracellular levels of H2O2. These findings provide a putative mechanistic explanation for the efficacy and selectivity of therapies such as ascorbic acid that rely on ROS-induced cell death for their anti-tumor properties.