Abstract A dynamic perfusion system was designed for cultivation an in vitro model comprising human intestinal colonoids, intestinal myofibroblasts, and monocyte-derived macrophages. The primary aim was to investigate the effect of therapeutic interventions on inflammatory bowel disease (IBD) using this bioengineered system. The approach involved creating a a colonoid epithelial monolayer on a scaffold lumen with myofibroblasts and macrophages in the subepithelial compartment, which was then integrated into a bioreactor enabling controlled perfusion mimicking the dynamic intestinal microenvironment to introduce inflammation and assess responses to traditional and innovative therapies. Inflammation induction in the model resulted in decreased epithelial integrity along with the upregulation of pro-inflammatory cytokines. The addition of myofibroblasts amplified the secretion of pro-inflammatory cytokines GCP-1, GM-CSF, and IL-1α, while concurrently reducing MCP-1 levels. The introduction of myofibroblasts contributed to an enhanced inflammatory response in the tissue. This system is poised to serve as a valuable tool for evaluating potential therapeutic strategies aimed at addressing IBD.

We establish an in vitro perfusion intestinal tissue bioreactor system tailored to study drug responses related to inflammatory bowel disease (IBD). The system includes key components including multiple human intestinal cell types (colonoids, myofibroblasts, and macrophages), a three-dimensional (3D) intestinal architecture, and fluid flow. Inclusion of myofibroblasts resulted in increased secretion of cytokines such as glypican-1 (GCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 1-α (IL-1α), whereas inclusion of macrophages resulted in increased secretion of monocyte chemoattractant proteins (MCPs) demonstrating a significant role of both stromal and immune cell types in intestinal inflammation. The system is responsive to drug treatments, as reflected in the reduction of pro-inflammatory cytokine production in tissue in some treatment scenarios. While future studies are needed to evaluate more nuanced responses in an IBD context, the present study demonstrates the ability to establish a 3D intestinal model with multiple relevant cell types and flow that is responsive to both inflammatory cues and various drug treatment options.

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γ-H2AX), which is an indicator of DNA double-strand breaks. While γ-H2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γ-H2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γ-H2AX immunostained images confirmed strong correlation between SLAM image features and γ-H2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Background: DNA damage response (DDR) is a fundamental mechanism for evaluating efficacy of cancer therapeutics, many of which target DNA or associated processes (e.g., etoposide: inhibits DNA synthesis, cisplatin: crosslinks DNA). Current techniques to evaluate DDR rely on immunostaining for gamma H2AX foci (γH2AX), which is an indicator of DNA double-strand break formation. While γH2AX immunostaining can provide a snapshot of DDR in fixed cellular and tissue samples, this method is technically cumbersome as: 1) temporal monitoring of DDR requires timepoint replicates; 2) extensive assay development efforts for 3D samples such as organoids, and 3) protocols for γH2AX immunostaining and its evaluation are time-consuming. The goal of this project is to reduce overall burden on assay duration and development in NSCLC organoids by leveraging label-free multiphoton imaging. Method: To predict DDR in 3D organoid samples, label-free multimodal multiphoton techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) imaging can provide intracellular information based on endogenous contrast mechanisms. SLAM makes use of a single laser to excite autofluorescence in multiple cellular components including FAD and NAD(P)H, enabling the calculation of the optical redox ratio, which estimates the equilibrium of electron donating versus accepting species in cells. Moreover, label-free, multiphoton systems enable imaging of live samples without the need to add labels or generate sacrificial samples and have improved image acquisition in 3D space over conventional confocal microscopy. Using SLAM, we propose to predict DDR in non-small cell lung cancer (NSCLC) organoids. NSCLC organoids recapitulate histological and genetic features of their derived patient tissue, retaining tumor variability. Results: Predictive modeling via gradient boost regressor between handcrafted SLAM image features and γH2AX immunostained images on weakly-paired images confirmed strong correlation between SLAM image features and γH2AX speckles/nuclei. Across multiple patient derived NSCLC organoid lines and multiple DNA targeting chemotherapeutics, the optical redox ratio was demonstrated to robustly predict DDR. Conclusion: Label-free imaging via SLAM can be used to predict DDR in live 3D NSCLC organoids with minimal sample handling and without the need to add labelled reporters, enabling quicker and less technically involved DDR assaying. Citation Format: Terrence T. Roh, Aneesh Alex, Janet E. Sorrells, Prasanna Chandramouleeswaran, Marina Marjanovic, Steve R. Hood, BanuPriya Sridharan, Stephen A. Boppart. Label-free multimodal multiphoton microscopy for predicting DNA damage response in patient derived non-small cell lung cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6613.

The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three-dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.

Animal models have delivered critical insights into mechanisms underlying the intestinal innate immune system; however, inherent differences exist between human and animal systems. To further understand the intestine innate immune system, there is a growing need for in vitro tissue model systems using human cells. A critical feature of in vitro cell and tissue models is the subepithelial environment, which contains additional cell types and includes 2D, microfluidic, organoid, and 3D tissue models. Where mouse models for the study of intestinal innate immune systems fall short, developments from in vitro models continue to grow in importance to aid efforts to understand this system in the context of disease and potential treatments.

Animal models have delivered critical insights into mechanisms underlying the intestinal innate immune system; however, inherent differences exist between human and animal systems. To further understand the intestine innate immune system, there is a growing need for in vitro tissue model systems using human cells. A critical feature of in vitro cell and tissue models is the subepithelial environment, which contains additional cell types and includes 2D, microfluidic, organoid, and 3D tissue models. Where mouse models for the study of intestinal innate immune systems fall short, developments from in vitro models continue to grow in importance to aid efforts to understand this system in the context of disease and potential treatments.

Designing biomimetic scaffolds with in vivo-like microenvironments using biomaterials is an essential component of successful tissue engineering approaches. The intestinal smooth muscle layers exhibit a complex tubular structure consisting of two concentric muscle layers in which the inner circular layer is orthogonally oriented to the outer longitudinal layer. Here, we present a three-dimensional (3D) bi-layered tubular scaffold based on flexible, mechanically robust and well aligned silk protein microfibers to mimic native human intestinal smooth muscle structure. The scaffolds were seeded with primary human intestinal smooth muscle cells to replicate human intestinal muscle tissues in vitro. Characterization of the tissue constructs revealed good biocompatibility and support for cell alignment and elongation in the different scaffold layers to enhance cell differentiation and functions. Furthermore, the engineered smooth muscle constructs supported oriented neurite outgrowth, a requisite step to achieve functional innervation. These results suggested these microfiber scaffolds as functional templates for in vitro regeneration of human intestinal smooth muscle systems. The scaffolding provides a crucial step toward engineering functional human intestinal tissue in vitro, as well as for the engineering of many other types of smooth muscles in terms of their similar phenotypes. Such utility may lead to a better understanding of smooth muscle associated diseases and treatments.

An in vitro model of intestinal epithelium with an immune component was bioengineered to mimic immunologic responses seen in inflammatory bowel disease. While intestinal immune phenomena can be modeled in transwells and 2D culture systems, 3D tissue models improve physiological relevance by providing a 3D substrate which enable migration of macrophages towards the epithelium. An intestinal epithelial layer comprised of non-transformed human colon organoid cells and a subepithelial layer laden with monocyte-derived macrophages was bioengineered to mimic native intestinal mucosa cell organization using spongy biomaterial scaffolds. Confluent monolayers with microvilli, a mucus layer, and infiltration of macrophages to the basal side of the epithelium were observed. Inflammation, induced by E. coli O111:B4 lipopolysaccharide and interferon γ resulted in morphological changes to the epithelium, resulting in ball-like structures, decreased epithelial coverage, and increased migration of macrophages to the epithelium. Analysis of cytokines present in the inflamed tissue model demonstrated significantly upregulated secretion of pro-inflammatory cytokines that are often associated with active inflammatory bowel disease, including CXCL10, IL-1β, IL-6, MCP-2, and MIP-1β. The macrophage layer enhanced epithelial and biochemical responses to inflammatory insult, and this new tissue system may be useful to study and develop potential therapies for inflammatory bowel disease.

There is a need for functional in vitro 3D human intestine systems that can bridge the gap between conventional cell culture studies and human trials. The successful engineering in vitro of human intestinal tissues relies on the use of the appropriate cell sources, biomimetic scaffolds, and 3D culture conditions to support vital organ functions. We previously established a compartmentalized scaffold consisting of a hollow space within a porous bulk matrix, in which a functional and physiologically relevant intestinal epithelium system was generated using intestinal cell lines. In this study, we adopt the 3D scaffold system for the cultivation of stem cell-derived human small intestinal enteriods (HIEs) to engineer an in vitro 3D model of a nonstransformed human small intestinal epithelium. Characterization of tissue properties revealed a mature HIE-derived epithelium displaying four major terminally differentiated epithelial cell types (enterocytes, Goblet cells, Paneth cells, enteroendocrine cells), with tight junction formation, microvilli polarization, digestive enzyme secretion, and low oxygen tension in the lumen. Moreover, the tissue model demonstrates significant antibacterial responses to E. coli infection, as evidenced by the significant upregulation of genes involved in the innate immune response. Importantly, many of these genes are activated in human patients with inflammatory bowel disease (IBD), implicating the potential application of the 3D stem-cell derived epithelium for the in vitro study of host-microbe-pathogen interplay and IBD pathogenesis.

The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments.

The present work investigated the osteogenic potential of injectable, dual thermally and chemically gelable composite hydrogels for mesenchymal stem cell (MSC) delivery in vitro and in vivo. Composite hydrogels comprising copolymer macromers of N-isopropylacrylamide were fabricated through the incorporation of gelatin microparticles (GMPs) as enzymatically digestible porogens and sites for cellular attachment. High and low polymer content hydrogels with and without GMP loading were shown to successfully encapsulate viable MSCs and maintain their survival over 28 days in vitro. GMP incorporation was also shown to modulate alkaline phosphatase production, but enhanced hydrogel mineralization along with higher polymer content even in the absence of cells. Moreover, the regenerative capacity of 2 mm thick hydrogels with GMPs only, MSCs only, or GMPs and MSCs was evaluated in vivo in an 8 mm rat critical size cranial defect for 4 and 12 weeks. GMP incorporation led to enhanced bony bridging and mineralization within the defect at each timepoint, and direct bone-implant contact as determined by microcomputed tomography and histological scoring, respectively. Encapsulation of both GMPs and MSCs enabled hydrogel degradation leading to significant tissue infiltration and osteoid formation. The results suggest that these injectable, dual-gelling cell-laden composite hydrogels can facilitate bone ingrowth and integration, warranting further investigation for bone tissue engineering.

In this study, we investigated the mineralization capacity and biocompatibility of injectable, dual-gelling hydrogels in a rat cranial defect as a function of hydrogel hydrophobicity from either the copolymerization of a hydrolyzable lactone ring or the hydrogel polymer content. The hydrogel system comprised a poly(N-isopropylacrylamide)-based thermogelling macromer (TGM) and a polyamidoamine crosslinker. The thermogelling macromer was copolymerized with (TGM/DBA) or without (TGM) a dimethyl-γ-butyrolactone acrylate (DBA)-containing lactone ring that modulated the lower critical solution temperature and thus, the hydrogel hydrophobicity, over time. Three hydrogel groups were examined: (1) 15wt.% TGM, (2) 15wt.% TGM/DBA, and (3) 20wt.% TGM/DBA. The hydrogels were implanted within an 8mm critical size rat cranial defect for 4 and 12weeks. Implants were harvested at each timepoint and analyzed for bone formation, hydrogel mineralization and tissue response using microcomputed tomography (microCT). Histology and fibrous capsule scoring showed a light inflammatory response at 4weeks that was mitigated by 12weeks for all groups. MicroCT scoring and bone volume quantification demonstrated a similar bone formation at 4weeks that was significantly increased for the more hydrophobic hydrogel formulations - 15wt.% TGM and 20wt.% TGM/DBA - from 4weeks to 12weeks. A complementary in vitro acellular mineralization study revealed that the hydrogels exhibited calcium binding properties in the presence of serum-containing media, which was modulated by the hydrogel hydrophobicity. The tailored mineralization capacity of these injectable, dual-gelling hydrogels with hydrolysis-dependent hydrophobicity presents an exciting property for their use in bone tissue engineering applications.

Abstract Acquired chemoresistance remains a major clinical issue in the management of advanced solid cancers. Initial response to cytotoxic chemotherapy is common, but certain patients progressively become unresponsive after multiple chemotherapy cycles. While causes of drug resistance are multiple and complex, here we approach this problem from a new angle: we studied whether repopulation of residual surviving cancer cells between chemotherapy cycles contributes to progressive chemoresistance. Currently the identity of repopulating cancer cells following chemotherapy is unknown, and the underlying molecular mechanisms that initiate tumor repopulation remain poorly understood. In the present study we use bladder cancer as a model and report that quiescent cancer stem cells (CSCs) are unexpectedly recruited to proliferate and repopulate residual tumors in response to chemotherapy-induced damage. This phenomenon is similar to how normal resident tissue stem cells mobilize to wound sites for tissue repair. We further investigate whether blockade of this wound-induced CSC repopulation can provide an innovative approach to abrogate chemoresistance. Previously we showed that cytokeratin 14 (CK14) marks the most primitive bladder cancer cells and abundance of CK14+ cancer cells in patients correlates with poor survival. Here, we followed the standard clinical chemotherapy regimen with gap periods to allow recovery of normal tissues between treatment cycles. While one cycle of gemcitabine and cisplatin effectively reduced tumor growth in vivo, a generalized expansion of CK14+ CSCs occurred in residual tumors during these gap periods between cycles. Further analysis revealed the induction of a “wound-response” gene signature in residual tumors and active recruitment of quiescent CSCs into proliferation in response to chemotherapy-induced damage. We demonstrated that prostaglandin E2 (PGE2) released by neighboring dying cancer cells could induce CSC expansion in a paracrine manner. This undesirable CSC expansion could be abrogated by a PGE2 neutralizing antibody and Celecoxib, an FDA approved COX2 inhibitor that blocks PGE2 signaling. In vivo administration of Celecoxib blocked the induction of “wound-response” gene signature and significantly attenuated progressive development of chemoresistance in xenograft tumors, including primary xenografts derived from a patient who failed chemotherapy.These results revealed a new mechanism by which CSCs contribute to therapeutic resistance via repopulating residual tumors between chemotherapy cycles. Repopulation was initiated by dying cells that induced wound response and recruitment of CSCs to repair chemotherapy-induced damages. Therapeutic intervention with Celecoxib effectively blocked this process and improved chemotherapeutic response in bladder tumors, supporting further validation in other solid cancers. Citation Format: Antonina V. Kurtova, Jing Xiao, Qianxing Mo, Senthil Pazhanisamy, Ross Krasnow, Seth P. Lerner, Fengju Chen, Terrence Roh, Erica Lay, Philip L. Ho, Keith S. Chan. Blocking wound-induced tumor repopulation between chemotherapy cycles as a novel approach to abrogate chemoresistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5470. doi:10.1158/1538-7445.AM2015-5470

Using human bladder cancer xenograft models, a new mechanism involving an active proliferative response of cancer stem cells to chemotherapy-induced damage is shown, driven by prostaglandin E2 (PGE2) release in a manner similar to PGE2-induced wound repair; pharmacological inhibition of the PGE2/COX2 axis by celecoxib attenuates chemoresistance, suggesting a possible adjunctive therapy for bladder carcinomas. Recent studies demonstrated that cancer stem cells (CSCs) have a survival advantage in response to chemotherapy. Here Antonina Kurtova and colleagues we investigate the idea that CSCs may actively proliferate in response to chemotherapy-induced damage in a manner analogous to the stem cell mobilization seen at wound sites during tissue repair. The authors show in bladder cancer models in mice, including patient-derived xenografts, that during successive cycles of chemotherapy, stem-cell-like CK14+ cells become enriched and repopulate the tumours in-between cycles. This is driven by prostaglandin E2 (PGE2) release from dying tumour cells, in a manner akin to PGE2-induced wound repair. Combination with an PGE2-neutralizing antibody attenuates chemoresistance and suggests a possible therapeutic strategy for bladder carcinomas. Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival1,2,3. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair4,5,6,7. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.