Maximizing the potential of aggressive mouse tumor models in preclinical drug testing

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT. First, genetically quiet, ATRT is a difficult tumor to target molecularly. Tumor cells need to divide to propagate tumor growth—intercepting the common crossroads in cell cycle progression is a feasible strategy. KIF11 is needed for bipolar spindle formation in metaphase. We identified KIF11 as a universal target of all ATRT-molecular-subtypes. Ispinesib, a KIF11-inhibitor, effectively inhibited tumor proliferation in all seven cell lines. A second challenge—a major challenge in preclinical drug testing in-vivo among aggressive tumor models, is the narrow therapeutic window to administer drugs within the limited murine lifespan. Our most aggressive ATRT tumor model was lethal in all mice within ~ 1 month of tumor implantation. Such short-surviving mouse models are difficult to employ for preclinical drug testing due to the narrow time window to administer drugs. To overcome this time restriction, we developed a clinical staging system which allowed physically-fit mice to continue treatment, in contrast to the conventional method of fixed drug-dose-duration regimen in preclinical testing which will not be feasible in such short-surviving mouse models. We validated this approach in a second embryonal brain tumor, medulloblastoma. This is a clinically relevant, cost-efficient approach in preclinical testing for cancer and non-cancer disease phenotypes. Widely used preclinical mouse models are not the most accurate and lack the aggressive tumor spectrum found within a single tumor type. Mice bearing the most aggressive tumor spectrum progress rapidly in the limited murine life-span, resulting in a narrow therapeutic window to administer drugs, and are thus difficult to employ in preclinical testing. Our approach overcomes this challenge. We discovered ispinesib is efficacious against two embryonal brain tumor types.


Fig. S1
A. Establishment of a large panel of patient-derived orthotopic xenograft (PDOX) mouse models using patient tumors and patient-tumor-derived cell lines of ATRT. CHLA-06 was the most aggressive phenotype in our panel of ATRT tumor models. A.

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Fig. S2
A. Schematic illustration of our Fitness-adapted approach to preclinical drug testing extracted from Fig. 1b. B. Table showing treatment days of ispinesib in Batch A animals (total 2 cycles were delivered), and Batch B animals (up to 7 cycles were delivered). Our clinical staging system was used to evaluate the health fitness of the mice daily (5 days/week) to determine further cycles of ispinesib. Ispinesib invivo dosing is 10mg/kg administered intraperitoneally every 4 days for three doses, with the treatment course repeated around day 21, depending on the mice fitness level. At Stage 3-4, we would monitor the health of the mice over the next few days before deciding if the mice remain stable/fit to receive additional cycles. A. Illustrated staging table on how the animals are staged. Note that not every animal displays the same pattern of progression in each parameter. The higher stage was used.

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Pictures demonstrating how additional challenge test (grip test) is performed. Mice were held at this angle to perform observations on the hind legs as well as the front legs, to determine how the mouse was moving its limbs in a daily examination. Holding the mouse too near the base of its tail will cause the examining hand to obstruct the view of the hind leg movements. This grip test manoeuvre allowed us to observe the mouse doing a front grip onto the grills, and back-kick on the hind legs.

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How Grip Test is Performed
STAGING   Stage 1 and 2 mice were in optimal health state. This data was used to analyse progression-free and overall survival as shown in Fig. 1h, Table showing the day of first symptoms observed, fulfilling Stage 3, 4 and 5 for Batch B animals. Stage 1 and 2 mice were in optimal health state. This data was used to analyse progression-free and overall survival as shown in Fig. 1h,   . Brains from ispinesib-treated animals (lower 2 panels) were more normal in appearance, in contrast to untreated control animals (upper panel) which were more necrotic in appearance. Similar findings were observed in Batch B animals (Fig. 1g).

Batch A animals
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#1197
Hematoxylin & eosin staining of each corresponding mouse brain sectioned in Batch A animals. Red arrows indicate tumor formation. Some brain specimens have poor quality due to overnight deaths of animals (#1197, #1311, #1200, #1301). #1301 did not appear to have any visible residual tumor, area indicated by red arrow demonstrated no tumor cells but a cavitation.  A. Additional ATRT cell lines (CHLA-05) were treated with ispinesib and evaluated for cell cycle changes (Fig. S7A) and apoptosis (Fig. S7B). Cell cycle analysis of CHLA-05 treated with ispinesib (17.7 nM) for 24 h by propidium iodide flow cytometry. CHLA-05 cells treated with ispinesib showed increased percentage in G2/M phase by 7.4% as compared to DMSO control. This increase was accompanied by the concomitant decrease in G1 phase by 19% and increase in sub-G1 phase by 11.8%. This result indicated that ispinesib induced G2/M arrest and subsequent cell death in CHLA-05 cells.

Ispinesib-Treated
A. DMSO   Corresponding CHLA-05 cells were treated with ispinesib and evaluated for cell death and apoptotic changes. Detection of apoptosis marker in CHLA-05 cells treated with ispinesib (17.7 nM) for 24 and 48 h. Percentage of secondary necrotic cells (AnV + /PI + cells) was higher after 48 hrs (23.5%). There was no difference in apoptotic cells (AnV + only) between DMSO control and ispinesib-treated cells.   Full length western blots for western blots used in main figures (Fig. 4d). Consistent brightness for each gel.