Anton Simeonov Ph.D. National Institute of Health

New Device Uses Patient Tumors as High-Throughput Screen Tool

Efforts to replace traditional cell culture–based testing of drug candidates with models that carry better predictive power, as relates to effect in human patients, have proliferated in recent years. These include 3D organotypic models, tissue printing platforms, and organs-on-chips. These two reports take the testing of new oncology drug candidates to the next level, that is, right into the human patient. In Klinghoffer et al., the team reports on a multi-needle-based device for injecting multiple drugs transcutaneously into discrete loci within the tumor in an actual patient (figure 1). Through extensive testing, the team demonstrated that up to eight drugs could be injected into mapped locations within the tumor, forming column-like tracks, and to remain acting locally, allowing the post-treatment analysis of drug efficacy and mechanism of action through the measurement of multiple biomarkers. The biomarker analysis was performed after 24–72 h of treatment by obtaining 4 μm histological sections every 2 mm along the injection column. The authors evaluated three xenograft models either with several oncology drugs or with the same drug dosed at different concentrations. Post-treatment analyses showed that the different drugs acted distinctly based on their previously established molecular mechanisms and that their action exhibited a dose–response effect. Moreover, it was shown that the local responses to the microinjected drugs could be used to predict the corresponding response to the systemically delivered substance, and the system was demonstrated to support a pilot screen of 97 drugs. Last but not least, the authors tested the injection device in human lymphoma patients where the procedure was found to be generally well-tolerated. In a companion paper, the Robert Langer's team provides an implantable microdevice containing a large number of drug-containing reservoirs that can be implanted inside the tumor. As a prototype, the team used a cylindrical device ∼0.8 mm thick for a delivery via biopsy needle (figure 2). By properly separating the drug-containing cavities along the cylindrical carrier, the team was able to control the crosstalk between adjacent drugs (either to eliminate it to study the individual agents or to retain it in order to test for advantageous drug–drug combinations). At this stage, the device contained 16 reservoirs, although the authors pointed out that further increase in the number would be possible. Further, changing the shape of the cavity, as well as manipulating the drug formulation, afforded control over the rate of delivery of drugs with different physicochemical properties. The two reports thus bring us one step closer to shifting the medium-to-high throughput testing of drug candidates from the laboratory to the patient.

The CIVO tumor microinjection platform. (A) The CIVO platform consists of a handheld array of up to eight needles capable of simultaneously penetrating subcutaneous tumors and delivering microdoses of candidate therapeutics. (B) For preclinical studies, tumors were grown as flank xenografts in immunocompromised mice and injected while mice were anesthetized. A chemically inert ITD was co-injected through each needle. (C) A representative example of the ITD signal from a tumor injected using a five-needle array visualized with a Xenogen In Vivo Imaging System (IVIS). (D) A longitudinal IVIS scan demonstrating the column-like distribution of the tracking dye signal from a single needle spanning the z axis of the tumor. (E) Tumor responses were assessed after resection of the tumor via histological staining of cross sections (4 mm thick) sampled at 2-mm intervals perpendicular to the injection column. (F) High resolution whole-slide scanning captured images of every cell from each 4-mm-thick tissue section. (G) A representative tumor response to microinjected drug at a single injection site. Nuclei, DAPI (4′,6-diamidino-2-phenylindole) (blue); ITD (green); a drug-specific biomarker (orange). (H) The resulting images were processed by a custom image analysis platform called CIVO Analyzer, which classifies the cells within each region of interest as biomarker-positive (green dots) or biomarker-negative (red dots).

In vivo drug sensitivity assay. (A) The device is implanted by needle directly into tissue, and drugs diffuse from device reservoirs into confined regions of tumor. Each region is assayed independently to assess the tumor-specific response to a given drug, such as apoptosis or growth arrest. A second biopsy needle selectively retrieves a small column of tissue that immediately surrounds and includes the device. This tissue contains the regions of drug diffusion and is used for determination of drug efficacy. (B) Three methods for precise control over the release profile of a given drug are demonstrated: reservoir opening size affects the rate of transport; the formulation of a drug in a polymer matrix (for example, PEG slows release of sunitinib versus free doxorubicin); and hydrophilic expansive hydrogels (to achieve rapid tissue uptake of highly insoluble drugs, such as lapatinib). Scale bars, 300 μm.

* Abstract from Science Translational Medicine 2015; Vol. 7, Issue 284, p. 284ra58

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

* Abstract from Science Translational Medicine 2015; Vol. 7, Issue 284, p. 284ra57

Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Science Translational Medicine titled "A technology platform to assess multiple cancer agents simultaneously within a patient's tumor." Authors of the paper are Klinghoffer RA, Bahrami SB, Hatton BA, Frazier JP, Moreno-Gonzalez A, Strand AD, Kerwin WS, Casalini JR, Thirstrup DJ, You S, Morris SM, Watts KL, Veiseh M, Grenley MO, Tretyak I, Dey J, Carleton M, Beirne E, Pedro KD, Ditzler SH, Girard EJ, Deckwerth TL, Bertout JA, Meleo KA, Filvaroff EH, Chopra R, Press OW, Olson JM. This article was also titled "An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors." Authors of the paper are Jonas O, Landry HM, Fuller JE, Santini JT Jr., Baselga J, Tepper RI, Cima MJ, Langer R.


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