A technology platform to assess multiple cancer agents simultaneously within a patient's tumor

Abstract

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 of 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.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. 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 μm 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-μm-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).

Fig. 2. CIVO microinjections result in spatially defined, non-overlapping drug distribution and quantifiable tumor responses

(A) Intratumoral vincristine (VCR) distribution was directly tracked by microinjection of 3H-labeled drug into discrete positions of xenografted Ramos lymphoma tumors. After injection, tumors were resected at 2, 8, 24, 48, and 72 hours, and sections were subjected to autoradiography and quantified as drug concentration as a function of distance. Data are averages ± SEM (n = 4 tumors per time point). (B) Parallel tumors were microinjected with the same amount of vincristine (1.5 μg) or a vehicle (Veh) control. Tumor responses at 24 hours at various distances from each injection site were visualized by staining for pHH3 or CC3. High-magnification hematoxylin and eosin (H&E) images are from the approximate regions designated by the red and blue boxes in the middle panels. The fraction of biomarker-positive cells was plotted as a function of radial distance from the injection site 72 hours after microinjection. Data are averages ± SEM (n = 6 tumors). *P < 0.05; **P < 0.001, Wald test. (C) Multiplexed microinjection of five different vincristine concentrations, each through a unique position within the array. Sections from resected tumors were stained and quantified. Data are averages ± SEM (n = 5 tumors at 24 hours, six tumors at 72 hours after microinjection).

Fig. 3. In vivo tumor responses are mechanism of action–specific and concentration-dependent

(A and B) Ramos lymphoma tumors were injected with arrays containing a vehicle control, mafosfamide (MAF), doxorubicin (DOX), vincristine, and prednisolone (PRED), each delivered from a distinct needle within the array, and tumors were resected 24 hours later. Tissue sections from multiple depths along the injection column were stained with H&E or antibodies recognizing γH2AX, pHH3, or CC3. (A) Representative whole-section images from resected tumors. (B) Individual injection sites from tumors. (C) Ramos lymphoma tumors were microinjected with varying concentrations of each drug, each concentration through a distinct needle within the array, and tumors resected 24 and 72 hours after microinjection. Tissue sections were stained with antibodies recognizing γH2AX, pHH3, or CC3, and the fraction of biomarker-positive cells was plotted as a function of radial distance from the injection site. Data are average responses across multiple tumors ± SEM (mafosfamide, n = 5; doxorubicin, n = 15; vincristine, n = 8; and prednisolone, n = 4).

Fig. 4. Localized responses to CIVO microinjection detect context-dependent drug-specific resistance and sensitivity, which correlate with long-term systemic outcomes

(A) Ramos and Res-Ramos tumors were microinjected with arrays containing vehicle control, doxorubicin, vincristine, mafosfamide, and prednisolone, and tumor responses were visualized by staining 4-μm sections from tumors resected 24 hours after microinjection (except for doxorubicin, shown at 72 hours). Tissue sections from multiple depths along the injection column were stained with antibodies recognizing γH2AX, pHH3, or CC3. The fraction of CC3+ cells was quantified using CIVO Analyzer, and the difference in response between the Ramos and Res-Ramos tumors was plotted as a function of radial distance from the injection site for each drug. Data are average differences in response across a minimum of three tumors 24 hours after microinjection (except for doxorubicin curves, which are from 72 hours) ± SEM. (B) Ramos and Res-Ramos tumor–bearing mice (n ≥ 10 per cohort) were treated systemically with saline (control), doxorubicin (3.3 mg/kg), vincristine (0.5 mg/kg), cyclophosphamide (20 mg/kg), or prednisone (0.2 mg/kg). Doxorubicin, vincristine, and cyclophosphamide were each administered intravenously once per week (q1w) for 4 weeks. Prednisone was administered orally (PO) 5 days per week for 4 weeks. Efficacy was assessed via tumor volume measurements, and data are means ± SEM. Kaplan-Meier survival curves were determined for the cyclophosphamide treatment groups (n = 10 per cohort); P value was determined by a log-rank test using vehicle control for the comparison. Tumor growth inhibition (TGI) was calculated (see Materials and Methods) at day 8 for each treatment condition compared to vehicle. P values were determined by a two-sided Student's t test. (C) Cultures of drug-naïve and doxorubicin-resistant Ramos (Res-Ramos) cell lines were seeded in drug-free medium or medium with various doxorubicin, vincristine, mafosfamide, or prednisolone concentrations. Cell viability was measured 72 hours after drug exposure. Data are representative from three separate experiments.

Fig. 5. CIVO predicts enhanced experimental mTOR inhibitor CC-115 efficacy in doxorubicin-resistant tumors

(A) Ramos (n = 4) and Res-Ramos (n = 3 at 2 hours; n = 5 at 24 hours) tumors were microinjected with arrays containing vehicle control, ridaforolimus, or CC-115. Tumor responses were visualized by staining for p4EBP1 and CC3. (B) The fraction of CC3+ cells was quantified using CIVO Analyzer and plotted as a function of radial distance from the injection site. Data are average responses 24 hours after micro-injection ± SEM. *P < 0.05, Wald test. (C) Cultures of drug-naïve (Ramos) and doxorubicin-resistant (Res-Ramos) cell lines were seeded in drug-free medium or medium with various concentrations of CC-115. Cell viability was tested using the PrestoBlue assay after 72 hours. IC50, median inhibitory concentration. (D and E) Ramos and Res-Ramos tumor–bearing mice (n = 6 per cohort) were treated systemically with saline (control) or CC-115 (5 mg/kg) administered PO daily for 25 days. (D) Efficacy was assessed via tumor volume measurements. Data are means ± SEM. TGI was calculated at day 25 for each treatment condition. *P < 0.05 compared to saline control, two-sided Student's t test. (E) Representative images of tumors from each treatment arm on day 25.

Fig. 6. Pilot clinical use of CIVO demonstrates feasibility of inducing localized responses in human lymphoma tumors

(A) Microinjection procedure in a patient with a palpable cancerous lymph node using an early CIVO clinical prototype. The patient's tumor was injected with microdoses of vincristine (1.5 μg) and ICG. Lymph nodes were resected 24 hours later, and tumor responses were visualized by staining 4-mm sections for CC3. (B) The fraction of CC3+ cells was quantified from one patient's tumor using CIVO Analyzer and plotted as a function of radial distance from the injection site. Data are average responses across three sections from multiple depths along the injection column ± SEM. (C) Tissue sections were stained for CC3, and the localized cell death response to vincristine was verified by evaluation of parallel H&E-stained sections.

Fig. 7. CIVO performance in canine lymphoma tumors demonstrates spatially restricted responses linked to drug mechanism

(A to C) CIVO microinjection procedure adapted for a clinic-like, veterinary setting (A and B) by incorporating the placement of a “guide needle” into the optimal injection site using ultrasound guidance, (C) which aligned the microinjection device to target all needles of the handheld device into the optimal location in the tumor, increasing injection success. (D) ITD signal seen beneath the skin with a blue flashlight and yellow filter glasses, from a canine tumor injected using an eight-needle CIVO array. (E) Cancerous lymph nodes in two dogs were microinjected with the same amount of vincristine (1.5 μg) or a vehicle control. Tumors were resected 24 hours after microinjection. The ITD (green) and tumor responses (CC3, red; pHH3, yellow) were visualized in 4-μm sections from multiple depths along the injection column. Individual injection sites are shown for each dog. The fraction of biomarker-positive cells for each dog was plotted as a function of radial distance from the injection site. Data are average responses across three sections at about 2-mm intervals along the injection column ± SEM.