Shreya Raghavan

Successful Implantation of Bioengineered, Intrinsically Innervated, Human Internal Anal Sphincter

BACKGROUND & AIMS To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.

BACKGROUND Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. METHODS We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. RESULTS Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). CONCLUSIONS Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays.

Successful implantation of physiologically functional bioengineered mouse internal anal sphincter

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.

Surgical implantation of a bioengineered internal anal sphincter

PURPOSE Fecal incontinence is a common disorder that can have devastating social and psychologic consequences. However, there are no long-term ideal solutions for such patients. Although loss of continence is multifactorial, the integrity of the internal anal sphincter (IAS) has particular significance. We previously described the development of 3-dimensional bioengineered constructs using isolated smooth muscle tissue from donor C57BL/6 IAS. We hypothesized that the bioengineered ring constructs would retain cellular viability and promote neovascularization upon implantation into a recipient mouse. METHODS Internal anal sphincter ring constructs were surgically implanted into the subcutaneous tissue of syngeneic C57BL/6 mice and treated with either fibroblastic growth factor 2 (0.26 microg daily) or saline controls using a microosmotic pump. Internal anal sphincter constructs were harvested after 25 days (range, 23-26 days) and assessed morphologically and for tissue viability. RESULT Gross morphology showed that there was no rejection. Rings showed muscle attachment to the back of the mouse with no sign of inflammation. Fibroblastic growth factor 2 infusion resulted in a significantly improved histologic score and muscle viability compared with the control group. CONCLUSIONS Three-dimensional bioengineered IAS rings can be successfully implanted into the subcutaneous tissue of recipient mice. The addition of fibroblastic growth factor 2 led to improved muscle viability, vascularity, and survival. This approach may become a feasible option for patients with fecal incontinence.

Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity

Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity.

Personalized Medicine Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids

Purpose: Chemoresistant ovarian cancers grow in suspension within the ascites fluid. To screen the effect of chemotherapeutics and biologics on resistant ovarian cancers with a personalized basis, we developed a 3D hanging drop spheroid platform. Experimental Design: We initiated spheroids with primary aldehyde dehydrogenase–positive (ALDH+) CD133+ ovarian cancer stem cells (OvCSC) from different patient samples and demonstrated that stem cell progeny from harvested spheroids was similar to the primary tumor. OvCSC spheroids were utilized to initiate tumors in immunodeficient mice. Drug responses to cisplatin and ALDH-targeting compound or JAK2 inhibitor determined whether the OvCSC population within the spheroids could be targeted. Cells that escaped therapy were isolated and used to initiate new spheroids and model tumor reemergence in a personalized manner. Results: OvCSC spheroids from different patients exhibited varying and personalized responses to chemotherapeutics. Xenografts were established from OvCSC spheroids, even with a single spheroid. Distinct responses to therapy were observed in distinct primary tumor xenografts similar to those observed in spheroids. Spheroids resistant to cisplatin/ALDH inhibitor therapy had persistent, albeit lower ALDH expression and complete loss of CD133 expression, whereas those resistant to cisplatin/JAK2 inhibitor therapy were enriched for ALDH+ cells. Conclusions: Our 3D hanging drop suspension platform can be used to propagate primary OvCSCs that represent individual patient tumors effectively by differentiating in vitro and initiating tumors in mice. Therefore, our platform can be used to study cancer stem cell biology and model tumor reemergence to identify new targeted therapeutics from an effective personalized medicine standpoint. Clin Cancer Res; 23(22); 6934–45. ©2017 AACR.

Self-renewal and CSCs in vitro enrichment: Growth as floating spheres

Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies.

Cellular Physiology of Gastrointestinal Smooth Muscle

Regulation of smooth muscle contraction/relaxation is essential for proper gastrointestinal function. Regulation of contraction occurs at the thick filament and thin filament levels. Thick filament (myosin)-mediated transient and rapid contraction is regulated through the activation of MLCK and MLC 20 phosphorylation. Sustained contraction is regulated by inhibition of MLCP through a Ca 2+ independent pathway. Thin filament (actin) regulation occurs by modulating the access to myosin binding domains. Phosphorylation of HSP27 and HSP20 are important modulators of contraction/relaxation. Under relaxed conditions, myosin binding domains on actin filaments are blocked. Contraction is associated with phosphorylation of actin binding proteins and the availability of myosin binding sites. Relaxation is regulated by receptor-ligand mediated or NO/CO-mediated activation of nucleotide cyclases. These result in activation of cyclic nucleotide-dependent kinases, PKA or PKG. Substrate phosphorylation by these kinases leads to relaxation, which appears to affect gastrointestinal smooth muscle at both the thick and thin filament levels.

Abstract AP22: CHEMORESISTANCE, TUMOR INITIATION AND TUMOR RE–EMERGENCE TRENDS IN MALIGNANT ASCITES–DERIVED OVARIAN CANCER STEM CELL SPHEROIDS

Due to the rarity of ovarian cancer stem cells (OvCSC; Conclusions: We have developed a hanging drop array model that incorporates primary ovarian cancer stem cells into a 3D spheroid, capable of initiating tumors in immuno-deficient mice. OvCSCs differentiate into heterogeneous progenies within spheroids, correlating with differing responses to conventional and stem cell targeting drug treatments, providing a platform for personalized therapeutics. We also developed a spheroid model that can mimic tumor re-emergence using cells that escape first line chemotherapy. This platform can be utilized to study spheroid and cancer stem cell biology, and model tumor re-emergence to identify new targeted therapeutics. This work was supported by the DOD OCRP Early Career Investigator Award W81XWH-13-1- 0134 (GM). Citation Format: Shreya Raghavan PhD, Maria Ward BS, Pooja Mehta MSc, Ronald J Buckanovich MD PhD, Geeta Mehta PhD. CHEMORESISTANCE, TUMOR INITIATION AND TUMOR RE–EMERGENCE TRENDS IN MALIGNANT ASCITES–DERIVED OVARIAN CANCER STEM CELL SPHEROIDS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP22.

Comparison of Angiogenic Growth Factors to Optimize Physiological Function of Implanted Tissue Engineered Internal Anal Sphincter (IAS)

Intestinal Peptide (VIP) or 8-bromo-cyclicAMP. Summary: Circular smooth muscle cells maintained viability when cultured on chitosan-coated tissue culture plates and preserved their normal spindle-like shape and expression of contractile smooth muscle markers. Bioengineered tissue constructs harvested from the scaffold maintained their contraction and relaxation responses compared to control tissue constructs that were not placed on the scaffold. Conclusion: Chitosan is non-toxic to colonic smooth muscle cells and provided a good matrix for their survival and maintenance of their contractile phenotype. The tissue constructs maintained the integrity of receptors for VIP and Acetylcholine, and the integrity of intracellular signaling pathways for contraction and relaxation. This is the first report of bioengineering colonic circular smooth muscle tissue constructs on biodegradable chitosan scaffolds. This study provides a basis for creating a functional colon with the possibility of building a longitudinal smooth muscle layer on top of the bioengineered circular layer. Supported by NIH1RC1DK087151.