Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

The landscape of drug sensitivity and resistance in sarcoma.

Sarcomas are a family of rare malignancies composed of over 100 distinct histological subtypes. The rarity of sarcoma poses significant challenges in conducting clinical trials to identify effective therapies, to the point that many rarer subtypes of sarcoma do not have standard-of-care treatment. Even for established regimens, there can be substantial heterogeneity in responses. Overall, novel, personalized approaches for identifying effective treatments are needed to improve patient out-comes. Patient-derived tumor organoids (PDTOs) are clinically relevant models representative of the physiological behavior of tumors across an array of malignancies. Here, we use PDTOs as a tool to better understand the biology of individual tumors and characterize the landscape of drug resistance and sensitivity in sarcoma. We collected n=194 specimens from n=126 sarcoma patients, spanning 24 distinct subtypes. We characterized PDTOs established from over 120 biopsy, resection, and metastasectomy samples. We leveraged our organoid high-throughput drug screening pipeline to test the efficacy of chemotherapeutics, targeted agents, and combination therapies, with results available within a week from tissue collection. Sarcoma PDTOs showed patient-specific growth characteristics and subtype-specific histopathology. Organoid sensitivity correlated with diagnostic subtype, patient age at diagnosis, lesion type, prior treatment history, and disease trajectory for a subset of the compounds screened. We found 90 biological pathways that were implicated in response to treatment of bone and soft tissue sarcoma organoids. By comparing functional responses of organoids and genetic features of the tumors, we show how PDTO drug screening can provide an orthogonal set of information to facilitate optimal drug selection, avoid ineffective therapies, and mirror patient outcomes in sarcoma. In aggregate, we were able to identify at least one effective FDA-approved or NCCN-recommended regimen for 59% of the specimens tested, providing an estimate of the proportion of immediately actionable information identified through our pipeline. Highlights: Standardized organoid culture preserve unique sarcoma histopathological featuresDrug screening on patient-derived sarcoma organoids provides sensitivity information that correlates with clinical features and yields actionable information for treatment guidanceHigh-throughput screenings provide orthogonal information to genetic sequencingSarcoma organoid response to treatment correlates with patient response to therapyLarge scale, functional precision medicine programs for rare cancers are feasible within a single institution.

Personalized chordoma organoids for drug discovery studies.

Chordomas are rare tumors of notochordal origin, most commonly arising in the sacrum or skull base. Chordomas are considered insensitive to conventional chemotherapy, and their rarity complicates running timely and adequately powered trials to identify effective treatments. Therefore, there is a need for discovery of novel therapeutic approaches. Patient-derived organoids can accelerate drug discovery and development studies and predict patient responses to therapy. In this proof-of-concept study, we successfully established organoids from seven chordoma tumor samples obtained from five patients presenting with tumors in different sites and stages of disease. The organoids recapitulated features of the original parent tumors and inter- as well as intrapatient heterogeneity. High-throughput screenings performed on the organoids highlighted targeted agents such as PI3K/mTOR, EGFR, and JAK2/STAT3 inhibitors among the most effective molecules. Pathway analysis underscored how the NF-κB and IGF-1R pathways are sensitive to perturbations and potential targets to pursue for combination therapy of chordoma.

Mineralisation of 14C-labelled polystyrene plastics by Penicillium variabile after ozonation pre-treatment.

Large amounts of polystyrene (PS), one of the most widely used plastics in the world, end up in the environment through industrial discharge and littering, becoming one of the major components of plastic debris. Such plastics, especially the small-sized microplastics and nanoplastics, have received increasing concerns in terms of their potential environmental risks. Feasible approaches for the degradation of PS in waste materials and in the environment are highly desirable. Physicochemical pretreatments of PS may be applied to enhance biological degradation. In the present study, we synthesized 14C-labelled PS polymers, either uniformly labelled on the ring ([U-ring-14C]-PS) or labelled at the ß-carbon position of the alkyl chain ([ß-14C]-PS), and investigated the mineralisation of the 14C-PS polymers by the fungus Penicillium variabile CCF3219 as well as the effect of ozonation as a physico-chemical pre-treatment on the mineralisation by the fungi. Biodegradation of the 14C-PS polymers was studied in liquid medium (pH 7.5, without additional carbon substrate) with P. variabile for 16 weeks. During the incubation time, 14CO2 was captured to calculate the mineralisation of 14C-PS and the remaining polymers were analysed by means of scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectrometry and gel-permeation chromatography (GPC). The results showed that the fungi mineralised both labelled polymers, and that the [U-ring-14C]-PS with a lower molecular weight led to a higher mineralisation rate. Ozonation pre-treatment strongly enhanced mineralisation of [ß-14C]-PS. SEM analysis showed that the surface of the ozonated [ß-14C]-PS became uneven and rough after the incubation, indicating an attack on the polymer by P. variabile. FT-IR analysis showed that ozonation generated carbonyl groups on the [ß-14C]-PS and the amount of the carbonyl groups decreased after incubation of the [ß-14C]-PS with P. variabile. GPC analysis showed that the molecular weights of the ozonated [ß-14C]-PS decreased after incubation. The present data suggest that ozonation pretreatment could be a potential approach for degradation of PS waste and remediation of PS-contaminated sites.

Development of an attached-growth process for the on-site bioremediation of an aquifer polluted by chlorinated solvents.

A procedure for the design of an aerobic cometabolic process for the on-site degradation of chlorinated solvents in a packed bed reactor was developed using groundwater from an aquifer contaminated by trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA). The work led to the selection of butane among five tested growth substrates, and to the development and characterization from the site's indigenous biomass of a suspended-cell consortium capable to degrade TCE (first order constant: 96 L gprotein(-1) day(-1) at 30 °C and 4.3 L gprotein(-1) day(-1) at 15 °C) with a 90 % mineralization of the organic chlorine. The consortium immobilization had strong effects on the butane and TCE degradation rates. The microbial community structure was slightly changed by a temperature shift from 30 to 15 °C, but remarkably affected by biomass adhesion. Given the higher TCE normalized degradation rate (0.59 day(-1) at 15 °C) and attached biomass concentration (0.13 gprotein Lbioreactor(-1) at 15 °C) attained, the porous ceramic carrier Biomax was selected as the best option for the packed bed reactor process. The low TeCA degradation rate exhibited by the developed consortium suggested the inclusion of a chemical pre-treatment based on the TeCA to TCE conversion via ß-elimination, a very fast reaction at alkaline pH. To the best of the authors' knowledge, this represents the first attempt to develop a procedure for the development of a packed bed reactor process for the aerobic cometabolism of chlorinated solvents.

Trichloroethylene aerobic cometabolism by suspended and immobilized butane-growing microbial consortia: a kinetic study.

A kinetic study of butane uptake and trichloroethylene (TCE) aerobic cometabolism was conducted by two suspended-cell (15 and 30°C) and two attached-cell (15 and 30°C) consortia obtained from the indigenous biomass of a TCE-contaminated aquifer. The shift from suspended to attached cells resulted in an increase of butane (15 and 30°C) and TCE (15°C) biodegradation rates, and a significant decrease of butane inhibition on TCE biodegradation. The TCE 15°C maximum specific biodegradation rate was equal to 0.011 mg(TCE ) mg(protein)(-1) d(-1) with suspended cells and 0.021 mg(TCE) mg(protein)(-1) d(-1) with attached cells. The type of mutual butane/TCE inhibition depended on temperature and biomass conditions. On the basis of a continuous-flow simulation, a packed-bed PFR inoculated with the 15 or 30°C attached-cell consortium could attain a 99.96% conversion of the studied site's average TCE concentration with a 0.4-0.5-day hydraulic residence time, with a low effect of temperature on the TCE degradation performances.