A microfluidic system for testing the responses of head and neck squamous cell carcinoma tissue biopsies to treatment with chemotherapy drugs

Tumors are heterogeneous masses of cells characterized pathologically by their size and spread. Their chaotic biology makes treatment of malignancies hard to generalize. We present a robust and reproducible glass microfluidic system, for the maintenance and “interrogation” of head and neck squamous cell carcinoma (HNSCC) tumor biopsies, which enables continuous media perfusion and waste removal, recreating in vivo laminar flow and diffusion-driven conditions. Primary HNSCC or metastatic lymph samples were subsequently treated with 5-fluorouracil and cisplatin, alone and in combination, and were monitored for viability and apoptotic biomarker release ‘off-chip’ over 7 days. The concentration of lactate dehydrogenase was initially high but rapidly dropped to minimally detectable levels in all tumor samples; conversely, effluent concentration of WST-1 (cell proliferation) increased over 7 days: both factors demonstrating cell viability. Addition of cell lysis reagent resulted in increased cell death and reduction in cell proliferation. An apoptotic biomarker, cytochrome c, was analyzed and all the treated samples showed higher levels than the control, with the combination therapy showing the greatest effect. Hematoxylin- and Eosin-stained sections from the biopsy, before and after maintenance, demonstrated the preservation of tissue architecture. This device offers a novel method of studying the tumor environment, and offers a pre-clinical model for creating personalized treatment regimens.

LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging

This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin, survivin splice variant (survivin 2B) and other anti-apoptotic proteins Bad, Bcl-2 and XIAP. Apart from the promising anti-cancer efficacy and the exceptional tumor targeting ability observed by multimodal imaging using near-infrared (NIR) imaging, magnetic resonance imaging (MRI) and computerized tomographic (CT) techniques, these NCs also maintained the immunomodulatory benefits of bLf as they were able to increase the RBC, hemoglobin, iron calcium and zinc levels in mice.