A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

Prospective prediction of clinical drug response in high-grade gliomas using an ex vivo 3D cell culture assay.

BACKGROUND: Clinical outcomes in high-grade glioma (HGG) have remained relatively unchanged over the last 3 decades with only modest increases in overall survival. Despite the validation of biomarkers to classify treatment response, most newly diagnosed (ND) patients receive the same treatment regimen. This study aimed to determine whether a prospective functional assay that provides a direct, live tumor cell-based drug response prediction specific for each patient could accurately predict clinical drug response prior to treatment. METHODS: A modified 3D cell culture assay was validated to establish baseline parameters including drug concentrations, timing, and reproducibility. Live tumor tissue from HGG patients were tested in the assay to establish response parameters. Clinical correlation was determined between prospective ex vivo response and clinical response in ND HGG patients enrolled in 3D-PREDICT (ClinicalTrials.gov Identifier: NCT03561207). Clinical case studies were examined for relapsed HGG patients enrolled on 3D-PREDICT, prospectively assayed for ex vivo drug response, and monitored for follow-up. RESULTS: Absent biomarker stratification, the test accurately predicted clinical response/nonresponse to temozolomide in 17/20 (85%, P = .007) ND patients within 7 days of their surgery, prior to treatment initiation. Test-predicted responders had a median overall survival post-surgery of 11.6 months compared to 5.9 months for test-predicted nonresponders (P = .0376). Case studies provided examples of the clinical utility of the assay predictions and their impact upon treatment decisions resulting in positive clinical outcomes. CONCLUSION: This study both validates the developed assay analytically and clinically and provides case studies of its implementation in clinical practice.

Single-cell dissection of cellular components and interactions shaping the tumor immune phenotypes in ovarian cancer.

Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.

PD-1/PD-L1 checkpoint inhibitors in combination with olaparib display antitumor activity in ovarian cancer patient-derived three-dimensional spheroid cultures.

Immune checkpoint inhibitors (ICIs) that target programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown modest activity as monotherapies for the treatment of ovarian cancer (OC). The rationale for using these therapies in combination with poly (ADP-ribose) polymerase inhibitors (PARP-Is) has been described, and their in vivo application will benefit from ex vivo platforms that aid in the prediction of patient response or resistance to therapy. This study examined the effectiveness of detecting patient-specific immune-related activity in OC using three-dimensional (3D) spheroids. Immune-related cell composition and PD-1/PD-L1 expression status were evaluated using cells dissociated from fresh OC tissue from two patients prior to and following 3D culture. The patient sample with the greatest increase in the proportion of PD-L1 + cells also possessed more activated cytotoxic T cells and mature DCs compared to the other patient sample. Upon cytokine stimulation, patient samples demonstrated increases in cytotoxic T cell activation and DC major histocompatibility complex (MHC) class-II expression. Pembrolizumab increased cytokine secretion, enhanced olaparib cytotoxicity, and reduced spheroid viability in a T cell-dependent manner. Furthermore, durvalumab and olaparib combination treatment increased cell death in a synergistic manner. This work demonstrates that immune cell activity and functional modulation can be accurately detected using our ex vivo 3D spheroid platform, and it presents evidence for their utility to demonstrate sensitivity to ICIs alone or in combination with PARP-Is in a preclinical setting.

Prospective Validation of an Ex Vivo, Patient-Derived 3D Spheroid Model for Response Predictions in Newly Diagnosed Ovarian Cancer.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.

The effect of sterilization on silk fibroin biomaterial properties.

The effects of common sterilization techniques on the physical and biological properties of lyophilized silk fibroin sponges are described. Sterile silk fibroin sponges were cast using a pre-sterilized silk fibroin solution under aseptic conditions or post-sterilized via autoclaving, γ radiation, dry heat, exposure to ethylene oxide, or hydrogen peroxide gas plasma. Low average molecular weight and low concentration silk fibroin solutions could be sterilized via autoclaving or filtration without significant loses of protein. However, autoclaving reduced the molecular weight distribution of the silk fibroin protein solution, and silk fibroin sponges cast from autoclaved silk fibroin were significantly stiffer compared to sponges cast from unsterilized or filtered silk fibroin. When silk fibroin sponges were sterilized post-casting, autoclaving increased scaffold stiffness, while decreasing scaffold degradation rate in vitro. In contrast, γ irradiation accelerated scaffold degradation rate. Exposure to ethylene oxide significantly decreased cell proliferation rate on silk fibroin sponges, which was rescued by leaching ethylene oxide into PBS prior to cell seeding.

Effects of Shiga toxin type 2 on a bioengineered three-dimensional model of human renal tissue.

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.

Cyst formation following disruption of intracellular calcium signaling.

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca(2+)) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca(2+)-activated Ca(2+) channel of the endoplasmic reticulum. We hypothesize that Ca(2+) signaling through PC2, or other intracellular Ca(2+) channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca(2+) signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca(2+) signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca(2+) transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca(2+) signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca(2+) signaling lead to ADPKD cysts.

Tissue-engineered kidney disease models.

Renal disease represents a major health problem that often results in end-stage renal failure necessitating dialysis and eventually transplantation. Historically these diseases have been studied with patient observation and screening, animal models, and two-dimensional cell culture. In this review, we focus on recent advances in tissue engineered kidney disease models that have the capacity to compensate for the limitations of traditional modalities. The cells and materials utilized to develop these models are discussed and tissue engineered models of polycystic kidney disease, drug-induced nephrotoxicity, and the glomerulus are examined in detail. The application of these models has the potential to direct future disease treatments and preclinical drug development.

Bioengineered 3D human kidney tissue, a platform for the determination of nephrotoxicity.

The staggering cost of bringing a drug to market coupled with the extremely high failure rate of prospective compounds in early phase clinical trials due to unexpected human toxicity makes it imperative that more relevant human models be developed to better predict drug toxicity. Drug-induced nephrotoxicity remains especially difficult to predict in both pre-clinical and clinical settings and is often undetected until patient hospitalization. Current pre-clinical methods of determining renal toxicity include 2D cell cultures and animal models, both of which are incapable of fully recapitulating the in vivo human response to drugs, contributing to the high failure rate upon clinical trials. We have bioengineered a 3D kidney tissue model using immortalized human renal cortical epithelial cells with kidney functions similar to that found in vivo. These 3D tissues were compared to 2D cells in terms of both acute (3 days) and chronic (2 weeks) toxicity induced by Cisplatin, Gentamicin, and Doxorubicin using both traditional LDH secretion and the pre-clinical biomarkers Kim-1 and NGAL as assessments of toxicity. The 3D tissues were more sensitive to drug-induced toxicity and, unlike the 2D cells, were capable of being used to monitor chronic toxicity due to repeat dosing. The inclusion of this tissue model in drug testing prior to the initiation of phase I clinical trials would allow for better prediction of the nephrotoxic effects of new drugs.

The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

The regulation of cystogenesis in a tissue engineered kidney disease system by abnormal matrix interactions.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) remains a major health care concern affecting several million patients worldwide and for which there is no specific treatment. We have employed a 3D tissue engineered disease-like system to emulate cystic structures in vitro and analyzed the extracellular matrix (ECM) interactions in it. The tissue system was developed by culturing normal or polycystin-1 silenced mouse Inner Medullary Collecting Duct (mIMCD) cells in ECM infused into 3D porous silk protein biomaterial scaffolds. In this system, the silk scaffolds provide slow degradation, biocompatibility, and maintain structure and transport for the 3D system, while the ECM molecules retain biological signaling. Using this 3D tissue system we provide evidence for an autocrine signaling loop involving abnormal matrix deposition (collagen type IV and laminin) and its integrin receptor subunit protein (Integrin-ß1) in Pkd1 silenced mIMCD cells. In addition, we report that abnormal pericystic ECM interactions between matrix molecules and integrin subunit proteins regulate the rate of cystogenesis in the disease system. Molecular signaling showed abnormalities in cyclin proteins and cell-cycle progression upon Pkd1 knockdown. Importantly, disruption of the abnormal matrix interactions by an additional knockdown (double-silencing) of integrin-ß1 in Pkd1 silenced cells reversed the abnormalities and reduced the rate of cystogenesis. Together, these findings indicate that abnormal matrix deposition and altered integrin profile distribution as observed in ADPKD and are critical in cystogenesis and should be considered a target for the development of therapeutics.

The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma.

The microenvironment plays a significant role in human cancer progression. However, the role of the tumor microenvironment in the epigenetic control of genes critical to cancer progression remains unclear. As transient E-cadherin expression is central to many stages of neoplasia and is sensitive to regulation by the microenvironment, we have studied if microenvironmental control of E-cadherin expression is linked to transient epigenetic regulation of its promoter, contributing to the unstable and reversible expression of E-cadherin seen during tumor progression. We used 3D, bioengineered human tissue constructs that mimic the complexity of their in vivo counterparts, to show that the tumor microenvironment can direct the re-expression of E-cadherin through the reversal of methylation-mediated silencing of its promoter. This loss of DNA methylation results from the induction of homotypic cell-cell interactions as cells undergo tissue organization. E-cadherin re-expression is associated with multiple epigenetic changes including altered methylation of a small number of CpGs, specific histone modifications, and control of miR-148a expression. These epigenetic changes may drive the plasticity of E-cadherin-mediated adhesion in different tissue microenvironments during tumor cell invasion and metastasis. Thus, we suggest that epigenetic regulation is a mechanism through which tumor cell colonization of metastatic sites occurs as E-cadherin-expressing cells arise from E-cadherin-deficient cells.

E-cadherin suppression directs cytoskeletal rearrangement and intraepithelial tumor cell migration in 3D human skin equivalents.

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, beta actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues.