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The use of three-dimensional (3D) bioprinting has remained at the forefront of tissue engineering and has recently been employed for generating bioprinted solid tumors to be used as cancer models to test therapeutics. In pediatrics, neural crest-derived tumors are the most common type of extracranial solid tumors. There are only a few tumor-specific therapies that directly target these tumors, and the lack of new therapies remains detrimental to improving the outcomes for these patients. The absence of more efficacious therapies for pediatric solid tumors, in general, may be due to the inability of the currently employed preclinical models to recapitulate the solid tumor phenotype. In this study, we utilized 3D bioprinting to generate neural crest-derived solid tumors. The bioprinted tumors consisted of cells from established cell lines and patient-derived xenograft tumors mixed with a 6% gelatin/1% sodium alginate bioink. The viability and morphology of the bioprints were analyzed via bioluminescence and immunohisto chemistry, respectively. We compared the bioprints to traditional two-dimensional (2D) cell culture under conditions such as hypoxia and therapeutics. We successfully produced viable neural crest-derived tumors that retained the histology and immunostaining characteristics of the original parent tumors. The bioprinted tumors propagated in culture and grew in orthotopic murine models. Furthermore, compared to cells grown in traditional 2D culture, the bioprinted tumors were resistant to hypoxia and chemotherapeutics, suggesting that the bioprints exhibited a phenotype that is consistent with that seen clinically in solid tumors, thus potentially making this model superior to traditional 2D culture for preclinical investigations. Future applications of this technology entail the potential to rapidly print pediatric solid tumors for use in high-throughput drug studies, expediting the identification of novel, individualized therapies.

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors.

Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Background: Glioblastoma (GBM) has a median survival of Methods: We used a Cytation5 Cell Imager to quantify MU by BTICs from NHAs in direct contact under normal (20%) or hypoxic oxygen tensions (5%). We obtained RT-sensitive (JX14P) patient-derived xenograft BTICs and generated a paired acquired-resistant (JX14P-RT) line. This was achieved by implanting primary tumors into flanks of athymic nude mice and serially treating with 6 fractions of 2Gy over 14 days for multiple passages until the median doubling time was halved. Cells were plated at a 1:1 ratio on Geltrex for 18h and exposed to 5% or 20% oxygen in serum-free media. NHAs were pre-labeled with a GFP-mitochondria tracker and BTICs were infected with a mCherry lentivirus. BTIC-MU was determined by quantifying double-positive cells in whole-well images. Viability was determined using CellTiterGlo, n = 4. Results: Time-lapse imaging revealed GFP-mitochondria transfer from NHAs to BTIC cells via TNTs stimulated by hypoxic conditions. We measured overall MU and cell viability in both BTIC lines. JX14P co-cultured with NHAs trended toward an increased MU (cell fraction) in hypoxia (Hyp) compared to Normoxia (Norm) (Norm = 32.61 ± 13, Hyp = 44.83 ± 5, P>0.167). JX14P exhibit higher cell viability (RLU) in hypoxia when mono- or co-cultured with NHAs (Mono: Norm = 10275 ± 901, Hyp = 12599 ± 579, P0.167). Compared to monoculture, JX14PRT exhibits higher cell viability in co-culture with NHAs under hypoxia (Mono: Norm = 9721 ± 255, Hyp = 10011 ± 1462, P>0.998, Co: Norm = 4928 ± 664, Hyp = 7805 ± 944, P Conclusions: RT-sensitive or -resistant BTICs cocultured with NHAs exhibit increased cell viability under acute hypoxia compared to Normoxia with a trend toward increased MU. Results indicate a potential protective effect following direct interaction with NHAs under hypoxia. We are further exploring metabolic changes in each cell type following mitochondrial exchange. Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Christian T. Stackhouse, Hasan Alrefai, Patricia H. Hicks, Taylor L. Schanel, Joshua C. Anderson, Andee M. Beierle, Christopher D. Willey. Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes [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 1273.

Background:Glioblastoma multiforme is one of the most devastating forms of brain cancer seen in children. Children diagnosed with high-grade gliomas have poor prognosis, and there remains a need for improvement in the standard of care. In order to improve the standard of care for this condition, it is necessary to develop in vitro models which better recapitulate the nature of the tumors. Three-dimensional bioprinting has proven to be an effective technique in creating microtumors using adult glioblastoma cell lines but remains unexplored in pediatric glioblastoma. As such, we sought to develop a protocol which would allow for the creation of three-dimensionally bioprinted microtumors using patient-derived pediatric glioblastoma cells. Methods:A Bio X three-dimensional bioprinter (Cellink) was utilized to create the microtumor constructs. The composition of the bioink was modified through varying concentrations of sodium alginate and gelatin, or gelatin-methacrylate (Gel-MA). The microtumor size and volume was optimized through the variation of extrusion time, nozzle pressure, and both nozzle and print bed temperature. The crosslinking technique was also optimized through the utilization of 2% calcium chloride for varying exposure times, and also a 405-nm light (for Gel-MA bioink) at varied light intensity, length of time of application to, and distance from, the printed structure. Bioprinted pediatric XD456 brain tumor initiating cell constructs were treated with both chemotherapeutics and radiation to determine the model’s capacity to respond to treatment. PamStation 12 (PamGene Inc.) Kinomic data and RNA-seq (Illumina) data were generated for the constructs. Imaging was performed using Cytation 5 imager (Agilent). Results:Fluorescent microscopy with Calcein AM and Sytox™ Orange (ThermoFisher), along with viability assessments via CellTiter-Glo®, demonstrated expected cytotoxic effects after exposure to 10 μM Cisplatin. Additionally, PamStation 12 Kinomic and RNA-seq data indicated a difference in kinomic signaling between the bioprinted constructs as compared to the XD456 cells cultured as neurospheres. Conclusions:The utilization of a three-dimensional bioprinter allows for the creation of microtumors using patient-derived pediatric glioblastoma cells. Future studies will investigate the microtumors’ capacity as a drug screening model. The model will also be developed to further recapitulate the native tumor microenvironment of pediatric glioblastoma. Citation Format: Andee M. Beierle, Taylor L. Schanel, Hasan Alrefai, Joshua C. Anderson, Patricia H. Hicks, Lauren C. Nassour, Christopher D. Willey. Developing pediatric glioblastoma microtumors with a three-dimensional bioprinter. [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 3568.

Background: Glioblastoma (GBM) is the most common malignant primary brain tumor with very poor prognosis. The current standard of care for GBM consists of maximal safe tumor resection, chemotherapy (temozolomide) and radiotherapy followed by adjuvant temozolomide and Tumor Treating Fields (TTFields). Unfortunately, GBM displays both inherent and acquired resistance to temozolomide and radiation and is incurable. While TTFields have been found to broadly induce endoplasmic reticulum stress, autophagy, cell membrane permeability, DNA replication stress, anti-mitotic and anti-migratory effects, it is unclear how well TTFields impact radiation resistant GBM. To better understand the cytotoxic benefit of TTFields on acquired radiation resistant GBM, we tested patient-derived xenograft (PDX) brain tumor initiating cells (BTICs) that had been selected for radiation resistance and compared them to their radiation sensitive parent. Methods: PDX were developed with subcutaneous implantation of patient GBM cells into immunodeficient (nu/nu) mice. Acquired radiation resistant PDXs were generated through in vivo selection (6-8 passages) using six fractions of 2 Gray of radiation over two weeks. PDX BTICs were cultured on laminin-coated cover slips under serum free conditions. Cytotoxicity was measured on both the two-dimensional sham control and two-dimensional TTFields treatment conditions over the course of 72 hours and analyzed with crystal violet staining for both imaging and absorbance quantification. PamStation 12 Kinomic data and RNA-seq (Illumina) data was generated for each PDX. Results: Crystal violet quantification and representative imaging indicated that TTFields inhibit cell growth in both parental and acquired radiation resistant PDX-derived BTICs. There is an observed TTFields sensitivity response from the acquired radiation resistant PDX, indicating that TTFields treatment does have a cytotoxic effect on acquired radiation resistant models (seen over four reproducible experiments). Baseline kinomic and transcriptomic differences between the PDX lines suggest potential TTFields sensitivity signatures. Conclusions: Ongoing studies are focused on validation of this cytotoxic effect on larger cohort of both acquired and inherent radiation resistant PDX cells. We plan to measure TTFields -treatment induced kinomic alterations to identify downstream signaling pathways associated with TTFields treatment response, as well as the development of acquired TTFields resistant GBM PDX cells. Citation Format: Taylor L. Schanel, Amber B. Jones, Andee M. Beierle, Hasan Alrefai, Lauren C. Nassour, Patricia H. Hicks, Joshua C. Anderson, Anita B. Hjelmeland, Christopher D. Willey. Establishing the cytotoxic benefit of Tumor Treating Fields on radiation sensitive and acquired radiation resistant glioblastoma patient derived xenograft pairs [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 1102.

Head and neck (HN) cancer recurrence is common, and selecting effective salvage systemic therapy remains difficult, particularly for oral cavity cancers. Developing a rapid, robust and predictive therapeutic testing system could support clinical decision-making and improve patient outcomes. We developed a Patient Therapy Evaluation System (PTES) that employs a three-dimensional (3D) fully human microtumor drug testing assay using tissue specimens collected during surgery of HN cancers. Remnant fresh tumor tissue from patients is dissociated into single-cell suspension that is embedded using a novel HuBiogel-cell encapsulation technology (3D microtumors). This high-throughput assay platform allows morphologic, functional, and molecular evaluations in parallel by real-time imaging, cell proliferation, and biomarker protocols. Microtumor viability, growth profiles, and drug screening data are captured at multiple time points up to 14 days. Our initial cohort included 57 patient specimens (53 squamous cell carcinomas, 1 verrucous carcinoma, 1 osteosarcoma, 1 ameloblastoma, and 1 non-cancerous lichenoid mucositis). HuBiogel-embedded tumor cells formed numerous multicellular colonies exhibiting distinct organization and growth patterns in 14-day microtumor cultures. Interestingly, epithelial, stromal and stem-cell like populations were preserved in HN microtumor models based on marker expression. Treatment with single (cisplatin, 5FU, docetaxel) drugs and their combinations resulted in tumor inhibitory responses (IC50) evaluated by CellTiter-Glo assay, and residual surviving cells were also recorded by Calcein-AM staining of 3D Microtumors. While patient-derived HN microtumors were produced with high success rates, factors associated with lower microtumor yield included smaller tumor specimens and low viability after dissociation. In conclusion, our new all human microtumor assay models replicating phenotypic, functional, and molecular properties ex vivo provide a potential theranostic tool for rapidly predicting drug sensitivity and improving treatment strategy for HN cancer patients. Citation Format: Christopher D. Willey, Yedeh P. Ying, Anthony B. Morlandt, Hope M. Amm, Patricia H. Hicks, Joshua C. Anderson, Andee M. Beierle, Carissa M. Thomas, Jason M. Warram, Jingsong Chen, Jeffrey A. Thomas, Katie Banko, Raj K. Singh. Head and neck cancer HuBiogel-embedded microtumor assay system for therapeutic efficacy testing of patient tumor specimens. [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 4554.

BACKGROUND/PURPOSE: Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. MATERIALS/METHODS: Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. RESULTS: The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. CONCLUSION: This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

Highlights • Rare, high grade neuroendocrine-like pediatric tumor has TYRO3 and ALK mutations. • Crizotinib downregulates STAT3 and ERK1/2 signaling in tumor. • In vivo analysis demonstrated crizotinib resistance. • Crizotinib resistant cells have upregulation of PDGFRß signaling. • PDGFRß inhibition overrides resistance., Patient-derived xenografts provide significant advantages over long-term passage cell lines when investigating efficacy of treatments for solid tumors. Our laboratory encountered a high-grade, metastatic, neuroendocrine-like tumor from a pediatric patient that presented with a unique genetic profile. In particular, mutations in TYRO3 and ALK were identified. We established a human patient-derived xenoline (PDX) of this tumor for use in the current study. We investigated the effect of crizotinib, a chemotherapeutic known to effectively target both TYRO3 and ALK mutations. Crizotinib effectively decreased viability, proliferation, growth, and the metastatic properties of the PDX tumor through downregulation of STAT3 signaling, but expression of PDGFRß was increased. Sunitinib is a small molecule inhibitor of PDGFRß and was studied in this PDX independently and in combination with crizotinib. Sunitinib alone decreased viability, proliferation, and growth in vitro and decreased tumor growth in vivo. In combination, sunitinib was able to overcome potential crizotinib-induced resistance through downregulation of ERK 1/2 activity and PDGFRß receptor expression; consequently, tumor growth was significantly decreased both in vitro and in vivo. Through the use of the PDX, it was possible to identify crizotinib as a less effective therapeutic for this tumor and suggest that targeting PDGFRß would be more effective. These findings may translate to other solid tumors that present with the same genetic mutations.

Simple Summary Children with high-risk neuroblastoma have limited therapeutic options poor survival rates. The neuroblastoma tumor microenvironment contributes the lack of response to many interventions so innovative methods are needed to study the effects of the tumor microenvironment on new therapies. In this manuscript, we review the current literature related to the components of the tumor microenvironment and to the use of three-dimensional printing as modality to study cancer. This review highlights the potential for using three-dimensional printing to create an artificial tumor microenvironment in the presence of neuroblastoma to provide improved preclinical testing of novel therapies. Abstract In the quest to advance neuroblastoma therapeutics, there is a need to have a deeper understanding of the tumor microenvironment (TME). From extracellular matrix proteins to tumor associated macrophages, the TME is a robust and diverse network functioning in symbiosis with the solid tumor. Herein, we review the major components of the TME including the extracellular matrix, cytokines, immune cells, and vasculature that support a more aggressive neuroblastoma phenotype and encumber current therapeutic interventions. Contemporary treatments for neuroblastoma are the result of traditional two-dimensional culture studies and in vivo models that have been translated to clinical trials. These pre-clinical studies are costly, time consuming, and neglect the study of cofounding factors such as the contributions of the TME. Three-dimensional (3D) bioprinting has become a novel approach to studying adult cancers and is just now incorporating portions of the TME and advancing to study pediatric solid. We review the methods of 3D bioprinting, how researchers have included TME pieces into the prints, and highlight present studies using neuroblastoma. Ultimately, incorporating the elements of the TME that affect neuroblastoma responses to therapy will improve the development of innovative and novel treatments. The use of 3D bioprinting to achieve this aim will prove useful in developing optimal therapies for children with neuroblastoma.

This paper details a passive, inductor-capacitor (LC) resonant sensor embedded in a commercial dressing for low-cost, contact-free monitoring of a wound; this would enable tracking of the healing process while keeping the site closed and sterile. Spiral LC resonators were fabricated from flexible, copper-coated polyimide and interrogated using external reader antennas connected to a two-port vector network analyzer; the forward transmission scattering parameter (S21) magnitude was collected, and the resonant frequency (MHz) and the peak-to-peak amplitude of the resonant feature were identified. These increase during the healing process as the permittivity and conductivity of the tissue change. The sensor was first tested on gelatin-based tissue-mimicking phantoms that simulate layers of muscle, blood, fat, and skin at varying phases of wound healing. Finite element modeling was also used to verify the empirical results based on the expected variations in dielectric properties of the tissue. The performance of the resonant sensors for in vivo applications was investigated by conducting animal studies using canine patients that presented with a natural wound as well as a controlled cohort of rat models with surgically administered wounds. Finally, transfer functions are presented that relate the resonant frequency to wound size using an exponential model (R2 = 0.58-0.96). The next steps in sensor design and fabrication as well as the reading platform to achieve the goal of a universal calibration curve are then discussed.

Uncoated resonant sensors are presented here for wireless monitoring of solvated ions, with progress made toward monitoring nitrates in agricultural runoff. The sensor, an open-circuit Archimedean coil, is wirelessly interrogated by a portable vector network analyzer (VNA) that monitors the scattering parameter response to varying ionic concentrations. The sensor response is defined in terms of the resonant frequency and the peak-to-peak amplitude of the transmission scattering parameter profile (|S21|). Potassium chloride (KCl) solutions with concentrations in the range of 100 nM – 4.58 M were tested on nine resonators having different length and pitch sizes to study the effect of sensor geometry on its response to ion concentration. The resonant sensors demonstrated an ion-specific response, caused by the variations in the relative permittivity of the solution, which was also a function of the resonator geometry. A lumped circuit model, which fit the experimental data well, confirms signal transduction via change in solution permittivity. Also, a ternary ionic mixture (composed of potassium nitrate (KNO3), ammonium nitrate (NH4NO3), and ammonium phosphate (NH4H2PO4)) response surface was constructed by testing 21 mixture variations on three different sensor geometries and the phase and magnitude of scattering parameters were monitored. It was determined that the orthogonal responses presented by resonant sensor arrays can be used for quantifying levels of target ions in ternary mixtures. Applications of these arrays include measuring the concentration of key ions in bioreactors, human sweat, and agricultural waters. Preliminary results are shown for calibration standards and real waterway samples in Iowa, USA.

A low-cost, passive resonant sensor was developed for wireless detection and measurement of ionic compounds. The sensor was fabricated as an open-circuit, Archimedean spiral composed of copper on a flexible, polyimide substrate. The sensor is interrogated by a two-loop antenna connected to a vector network analyzer (VNA) to monitor the scattering parameter response of the sensor when exposed to varying ionic concentrations. The sensor response was defined in terms of the resonant frequency and the peak-to-peak amplitude of the transmission scattering parameter profile (|S21|). Potassium chloride (KCl) solutions with concentrations in the range of 100 nM – 4.58 M were tested on nine resonators having different length and pitch sizes to study the effect of sensor geometry on its response to ion concentration. The resonant sensors demonstrated an ion-specific response, caused by the variations in the relative permittivity of the solution, which was also a function of the resonator geometry. A lumped circuit model, which fit the experimental data well, confirms signal transduction via change in solution permittivity. Also, a ternary ionic mixture (composed of potassium nitrate (KNO3), ammonium nitrate (NH4NO3), and ammonium phosphate (NH4H2PO4)) response surface was constructed by testing 21 mixture variations on three different sensor geometries and the phase and magnitude of scattering parameters were monitored. It was determined that the orthogonal responses presented by resonant sensor arrays can be used for quantifying levels of target ions in ternary mixtures. Applications of these arrays include measuring the concentration of key ions in bioreactors, human sweat, and agricultural waters.