Impact of SCID mouse gender on tumorigenicity, xenograft growth and drug-response in a large panel of orthotopic PDX models of pediatric brain tumors

Abstract

Brain tumor is the leading cause of cancer related death in children. Clinically relevant animals are critical for new therapy development. To address the potential impact of animal gender on tumorigenicity rate, xenograft growth and in vivo drug responses, we retrospectively analyzed 99 of our established patient derived orthotopic xenograft mouse models (orthotopic PDX or PDOX). From 27 patient tumors, including 5 glioblastomas (GBMs), 11 medulloblastomas (MBs), 4 ependymomas (EPNs), 4 atypical teratoid/rhabdoid tumors (ATRTs) and 3 diffuse intrinsic pontine gliomas (DIPGs), that were directly implanted into matching locations in the brains of approximately equal numbers of male and female animals (n = 310) in age-matched (within 2-week age-difference) SCID mice, the tumor formation rate was 50.6 ± 21.5% in male and 52.7 ± 23.5% in female mice with animal survival times of 192.6 ± 31.7 days in male and 173.9 ± 34.5 days in female mice (P = 0.46) regardless of pathological diagnosis. Once established, PDOX tumors were serially subtransplanted for up to VII passage. Analysis of 1,595 mice from 59 PDOX models (18 GBMs, 18 MBs, 5 ATRTs, 6 EPNs, 7 DIPGs and 5 PENTs) during passage II and VII revealed similar tumor take rates of the 6 different tumor types between male (85.4 ± 15.5%) and female mice (84.7 ± 15.2%) (P = 0.74), and animal survival times were 96.7 ± 23.3 days in male mice and 99.7 ± 20 days in female (P = 0.25). A total of 284 mice from 7 GBM, 2 MB, 1 ATRT, 1 EPN, 2 DIPG and 1 PNET were treated with a series of standard and investigational drugs/compounds. The overall survival times were 106.9 ± 25.7 days in male mice, and 110.9 ± 31.8 days in female mice (P = 0.41), similar results were observed when different types/models were analyzed separately. In conclusion, our data demonstrated that the gender of SCID mice did not have a major impact on animal model development nor drug responses in vivo, and SCID mice of both genders are appropriate for use.

1. Introduction

Brain tumor is the most common childhood cancer other than leukemia and remain the leading cause of cancer-related death in children [1-7]. Despite recent advances of imaging diagnosis, surgical techniques and continually improved chemo-, radiation- and many other adjuvant therapies, the overall survival in many types of malignant pediatric brain tumors has not improved significantly. The five-year survival rate in children with high grade glioma is still <25%, and nearly all patients with diffuse intrinsic pontine glioma (DIPG) succumb to this disease within 9–12 months [8]. Even among brain tumor survivors, many patients are left with long-term cognitive and/or neuroendocrine sequalae [9-13]. Clinically relevant and molecularly accurate animal models are therefore needed to understand tumor biology and to develop novel and less-toxic therapies.

Direct implantation of patient tumor cells into the matching locations in the brains of SCID mice, i.e. cerebral tumor into mouse cerebra, cerebellar tumors to mouse cerebella and brain stem tumors into mouse brain stem, has led to the successful establishment of multiple pediatric brain tumor models [14-23]. Detailed characterization has also shown faithful replication of key histopathology, invasion and metastasis, as well as genetic features of the original tumors in these patient derived orthotopic xenograft (orthotopic PDX or PDOX) mouse models [14-17,20,24-26]. Many of them have been successfully utilized for understanding tumor biology and testing various new therapies [27-32].

While it is well established that host gender is important for cancers predominantly or exclusively occur in female (such as breast cancer) or male (such as prostate cancer) patients, recent studies have further shown that the gender of patient can have significant impact on tumor biology and drug responses in cancers that affect both female and male patients as well. In GBMs, for example, a recent study showed that standard therapy is more effective in female patients compared with male patients and cell cycle and integrin signaling were shown to be critical determinants of survival for male and female patients, respectively [33]. An analysis of GBM patient data also showed that proliferating monocytic myeloid-derived suppressor cells (mMDSC) that block antitumor immunity MDSCs were predominant in male tumors and that a high granulocytic myeloid-derived suppressor cell (gMDSC)/IL1β gene signature correlated with poor prognosis in female patients [34]. A large-scale glioma genome-wide association studies (GWAS) even identified sex-specific risk locus [35].

Despite widespread use of patient-derived xenograft animal models, the impact of host gender (e.g. male v. s female mice) on xenograft tumor growth and therapy responsiveness has not been well established. While male animals were frequently used for acute genotoxicity [36], female mice are primarily favored for preclinical drug testing [37-40] because male mice from separate litters usually do not get along in the cage and some mice are lost to injuries during the experiments. Recent studies, however, showed that tumor biology and drug responses in female mice were not always identical to tumors in male animals [34,41]. Since the genes and signaling pathways targeted by different treatment as well as drug metabolism and toxicity can potentially be influenced by the sex hormone and/or gender-related host differences, it is important to systematically analyze the similarities and differences between female and male immunodeficient animals on the xenograft tumor formation rate and in vivo responsiveness toward standard therapies and various investigational anti-cancer agents using a large panel of PDOX models. Indeed, the issue of gender-drug effect in vivo has gained prominence in recent years and National Institute of Health (NIH) announced the requirement for the use of both male and female animals in preclinical testing of drug safety and efficacy [42].

To evaluate the impact of animal sex difference in PDOX models of pediatric brain tumors, we retrospectively analyzed our panel of 99 PDOX models in which childhood brain tumors were implanted with approximately equal numbers of male and female SCID mice, on their tumorigenicity rate and xenograft growth changes with primary patient tumors and during serial in vivo subtransplantations as well as their responses toward standard and investigational anti-cancer agents. Our aim is to provide a large series of experimental data to inform future selection of animal genders during model development and preclinical drug testing.

2. Materials and Methods

2.1. Tumor tissues

Fresh surgical tumor tissues were collected in patients undergoing surgery at Texas Children’s Hospital, including patients with glioblastoma (GBM), medulloblastoma (MB), ependymoma (EPN), atypical teratoid rhabdoid tumor (ATRT) and primitive neuroectodermal tumor (PENT). Samples from consented autopsied diffuse intrinsic pontine glioma (DIPG) were also collected. Signed informed consent was obtained from the patient or legal guardian prior to sample acquisition in accordance with Institutional Review Board (IRB) policy as we described previously [14,15,17,20].

2.2. Patient-derived orthotopic xenograft mouse models

Tumor tissues were washed and minced with fine scissors into small fragments. Single cells and small clumps (3–5 cells per clump) of tumor cells were collected with a 40 μm cell strainer, then resuspended in Dulbecco’s modified Eagle’s medium (DMEM) to achieve a final concentration of 0.5 × 10⁸ live cells per ml, as assessed by trypan blue staining, and transferred to animal facility on ice. The SCID mice were bred and housed in a specific pathogen free animal facility at Texas Children’s Hospital. All the experiments were conducted using an Institutional Animal Care and Use Committee approved protocol. Surgical transplantation of tumor cells into mouse cerebellum, cerebrum and brain stem, usually completed within 60 min of tumor removal, was performed as described previously [14]. Both male and female mice, aged 4–6 weeks, were anesthetized with sodium pentobarbital (50 mg/kg, i. p. Injections) or isoflurane . Tumor cells (1 × 10⁵) were suspended in 2 μL of culture medium and injected into the right cerebellum (1 mm to the right of the midline, 1 mm posterior to the lamboidal suture, and 3 mm deep), or right cerebral hemisphere (1 mm to the right of the midline, 1.5 mm anterior to the lamboidal suture and 3 mm deep) or brain stem (1 mm to the right of the midline, 1 mm posterior to the lamboidal suture, and 5.2 mm deep) via a 10 μL 26-gauge Hamilton Gastight 1701 syringe needle (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com). The animals were then monitored daily for development of neurological deficits, at which time they were euthanized, and their brains removed for histopathologic examination. Those mice without any neurological deficit after 12 months were euthanized and examined for tumor development.

2.3. Serial subtransplantation of xenografts in vivo in mouse brains

Whole brains of donor mice were aseptically removed, coronal cut into halves, and transferred back to the tissue culture laboratory. Xenograft tumors were then dissected, mechanically dissociated into cell suspensions, and injected into the brains of recipient SCID mice as described above [14,15,17,20].

2.4. In vivo treatment for PDOX model

Two weeks after tumor cell implantation, mice were administered standard and investigational drugs/compounds, including radiation, oncolytic virus (SVV-001) [43], ABT888, MLN8237, TMZ, Flavopiridol, VPA, SAHA, Cisplatin and Panobinostat [25] as showed in Table 2. To determine any survival benefits from drug treatment, the mice were monitored daily until they developed signs of neurologic deficit or became moribund, at which time they were euthanized, and their brains were removed for analysis.

Table 2.

Tumorigenicity of established PDOX models during serial subransplantation between male and female SCID mice.

Tumor type	Model number	Total mouse number	Passages
GBM	18 (34a)	467	II-VII
MB	18 (33)	523	II-V
ATRT	5 (10)	120	II-V
EPN	6 (8)	91	II-V
DIPG	7 (20)	310	II-VII
PNET	5 (7)	84	II-V
Total	59 (112)	1595	II-VII

^aModel number including different passages in vivo.

2.5. Statistical analysis

Comparison of the numbers of PDOX models between male and female mice were performed with the Student t-test, Animal survival times were evaluated by log-rank analysis followed by pairwise multiple comparison procedures using the Holm-Sidak method with SigmaPlot 13 (Systat Software). P values < 0.05 are considered significant.

3. Results

3.1. Development of PDOX mouse models of pediatric brain tumors

As detailed in the Materials and Methods section, patient tumor tissues obtained from surgery or autopsy were mechanically dissociated and injected into the matching locations in the brains of SCID mice at 1 × 10⁵ cells for all intra-cerebral (IC) and intra-cerebellar (ICb) tumors and 5 × 10⁴ for intra-brain stem tumors (Fig. 1A and B) [44]. All the animals were carefully monitored, and the mice that developed signs of neurological deficit or became moribund were euthanized following our Institutional Animal Care and Use Committee (IACUC) approved protocol. Gross examination usually revealed the enlargement of the mouse brains with frequent visualization of tumor growth, particularly intra-cerebral or intra-cerebellar tumors (Fig. 1C). Serial section of paraffin embedded whole mouse brains was also performed to confirm tumor formation (Fig. 1C).

Fig. 1.

Xenograft tumor of PDOX in the mouse brain. (A, B) Images illustrating intra-cerebral (IC) and intra-cerebellar (ICb) and intra-brain stem (IBs) injections of pediatric brain tumor cells in the brains of SCID mice. 1 × 10⁵ brain tumor cells was injected into SCID mouse within 60 min of tumor resection into cerebellum, cerebrum or brain stem to establish the medulloblastoma (MB), glioblastoma (GBM), ependymoma (EPN), diffuse intrinsic pontine glioma (DIPG), Atypical Teratoid Rhabdoid Tumor (ATRT) and primitive neuroectodermal tumor (PNET) models. (C) Gross appearance of H&E-stained mouse brain with huge xenograft tumors.

3.2. The impact of animal gender on tumorigenicity and xenograft growth at passage I

To determine if animal gender affected xenograft forming capabilities of patient tumors, we retrospectively analyzed gender distribution in 99 PDOX models in which at least 1 mouse developed xenograft tumor and identified 27 models of which the human tumor cells were implanted into both male and female mice that were age-matched (within 2 weeks age-difference) (Table 1). The remaining 72 models were excluded because the tumors were injected to SCID mice of single gender as the gender differences were not considered an issue when human brain tumors were implanted several years earlier in our laboratory and the selection of mice were primarily based on the availability of young (4–6 week) animals (to mimic the developing brain of childhood brain tumors). Among 27 PDOX models, there were 5 GBMs, 11 MBs, 4 EPNs, 4 ATRTs and 3 DIPGs (Supplemental Table 1) with a comparable average number of male ranging from 3.8 ± 1 (in EPNs) to 10.3 ± 4.7 (in DIPGs), and of female mice from 4.2 ± 0.8 (in GBMs) to 8.7 ± 3.2 (in DIPGs). Since the overall number differences between male and female mice in all the five models were not significant (P > 0.05) (Fig. 2A and Table S1), we proceed to compare the tumorigenicity in these models.

Table 1.

Tumorigenicity of pediatric brain tumors in male and female SCID mice.

Model name	Formed tumor in both gender		Formed tumor in single gender
	Model number	Mouse number	Model number	Mouse number
GBM	5	48	24	296
MB	11	124	39	411
ATRT	4	47	7	93
EPN	4	34	18	169
DIPG	3	57	11	217
Subtotal	27	310	99	1186

¹GBM: Glioblastoma Multiforme.

²MB: Medulloblastoma.

³ATRT: Atypical Teratoid Rhabdoid Tumor.

⁴EPN: Anaplastic Ependymoma.

⁵DIPG: Brain Stem Glioma.

Fig. 2.

The impact of animal gender on tumorigenicity and survival time of PDOX models of primary passage in vivo. From total 1186 mice of passage I (P–I), the animal number (A), tumor formation rate (B, C) and survival times (D, E) of male mice bearing tumors were compared with that of female mice from 5 GBMs, 11 MBs, 4 ATRTs, 4 EPNs and 3 DIPGs models when the mice reached the endpoint. P > 0.05 means no difference between male and female mice.

Tumor forming rate in the 5 GBM models were 78.2 ± 33.4% in male and 84 ± 35.8% in female mice. The difference was not significant (P = 0.18). Similar results were found in MBs (78.4 ± 27.9% in male vs. 75 ± 33.2% in female) (P = 0.28); ATRTs (61.7 ± 32.8% in male vs. 70.6 ± 25.5% in female) (P = 0.26); EPNs (47.5 ± 14.5% in male: 53.8 ± 17% in female) (P = 0.39) and DIPGs (37.9 ± 20.3% in male: 22.7 ± 15.5% in female) (P = 0.24) (Fig. 2B). When all the male and female mice were grouped together regardless of the tumor type, the tumor take rate was 50.6 ± 21.5% in male and 52.7 ± 23.5% in female, not statistically significant (P = 0.92) (Fig. 2C).

We next examined if there were any differences of tumor growth between male and female mice by comparing animal survival times of tumor-bearing mice. In the 5 GBM models, the animal survival times was 126 ± 21.8 days in male and 112.6 ± 20.5 days in female mice (P = 0.31) and in the 11 MB models, 183.5 ± 40.3 days in male and 191.1 ± 43.2 days in female mice (P = 0.6). Similarly findings were revealed in the 4 ATRT models (141.4 ± 20.8 days in male and 129.6 ± 15.7 days in female mice) (P = 0.43), 4 EPN models (225.5 ± 75.7 days in male and 255.4 ± 90.3 days in female mice) (P = 0.58), and 3 DIPG models (286.8 ± 31.7 days in male and 180.8 ± 37.5 days in female mice) (P = 0.52) (Fig. 2D). When combined, the survival times of male mice of the 5 types of pediatric brain tumors was 192.6 ± 31.7 days, similar to that in female mice (173.9 ± 34.5 days) (P = 0.46) (Fig. 2E). Altogether, our data did not reveal significant differences in tumor take rate and tumor growth between male and female mice during PDOX model establishment from patient tumor tissues of 5 types of pediatric brain tumors.

3.3. The impact of animal gender on tumor take and growth during serial subtransplantations

Similar to human brain tumors, animals bearing malignant tumor xenografts in their brain eventually all died of the disease. Serial subtransplantation, in which PDOX tumors harvested from donor mice were implanted serially in recipient mice, provided a critical means for sustained supply of PDOX models. It is therefore very important to examine if animal gender would affect tumor take and tumor growth during serial in vivo subtransplantations. From our database, we identified 59 PDOX models (with a total of 1595 mice), including 18 GBMs, 18 MBs, 5 ATRTs, 6 EPNs, 7 DIPGs and 5 PNETs (note: PNET was discontinued in the 2016 WHO classification), that were implanted with approximately equal numbers of male and female animals during serial subtransplantation from passage II up to passage VII. Among these PDOX models, the average number of male animals ranged from 5.1 ± 2 (in EPNs) to 8.2 ± 4.8 (in MBs), and of female mice from 6.3 ± 3 (in EPNs) to 7.4 ± 3.1 (in DIPGs). Since the overall number differences between male and female mice in all the five models were not significant (P > 0.05) (Table 2 and Fig. 3A), we proceeded to compare the tumorigenicity in these models.

Fig. 3.

The impact of animal gender on tumorigenicity and survival time of subtransplanted PDOX models in vivo. From total 1595 mice of passage II (P-II) to passage VII (P-VII), the animal number (A), tumor formation rate (B, C) and survival times (D, E) of male mice bearing tumors were compared with that of female mice from 18 GBMs, 18 MBs, 5 ATRTs, 6 EPNs, 7 DIPGs and 5 PNETs models when the mice reached the endpoint; P > 0.05 means no difference between male and female mice.

Tumor forming rate in the 18 GBM models were 93.9 ± 12.8% in male and 96.5 ± 10.1% in female mice. The difference was not significant (P = 0.33). Similar results were found in MBs (93.4 ± 11.7% in male vs. 94.9 ± 9.4% in female) (P = 0.18); ATRTs (94.2 ± 13.5% in male vs. 94.4 ± 9.8% in female) (P = 0.23); EPNs (83.1 ± 27.6% in male and 72.5 ± 31.5% in female) (P = 0.45); DIPGs (48.1 ± 27.6% in male and 52.8 ± 24.9% in female) (P = 0.9) and PNETs (100 ± 0% in male vs. 96.8 ± 5.5% in female) (P = 0.16) (Fig. 3B). When all the male and female mice were grouped together regardless of the tumor type, the tumor take rate was 85.4 ± 15.5% in male and 85 ± 15.2% in female, not statistically significant (P = 0.74) (Fig. 3C).

With the similar tumor take rate and animal number per group, we next compared the animal survival times of tumor bearing mice. In GBM models, it was 84.4 ± 18.3 days in male mice and 77.5 ± 15.7 days in female mice (P = 0.92); whereas in MB models, 119 ± 34.4 days in male and 124.2 ± 32.4 days in female mice (P = 0.89). Similar results were found in ATRT models (94.5 ± 21.6 days in male mice vs 96.9 ± 15.2 days in female, P = 0.06); in EPN models (129.7 ± 23.9 days vs 138.9 ± 24.9 days, P = 0.62); in DIPG models (74.9 ± 28.4 days vs and 82 ± 21.9 days, P = 0.81); in PNET models (77.8 ± 13.4 days vs 79.3 ± 9.8 days, P = 0.76) (Fig. 3D). When combined, the survival times of tumor-bearing mice was 97 ± 23.3% days in male and 100 ± 20% days in female (P = 0.25) (Fig. 3E), indicating that the SCID mice of both genders are appropriate for tumorigenicity and xenograft growth in PDOX model development.

3.4. The impact of animal gender on in vivo drug responses in preclinical drug testing

A total of 284 mice from 7 GBM, 2 MB, 1 ATRT, 1 EPN, 2 DIPG and 1 PNET were treated with a series of standard and investigational drugs/compounds, including radiation, oncolytic virus (SVV-001), ABT888, MLN8237, temozolomide (TMZ), Flavopiridol, valproic acid (VPA), SAHA, Echinomycin, MCB613, Cisplatin, Panobinostat (Table 3A). The scheme of administration was described as in Table 3B. The overall survival times were 106.93 ± 25.7 days in male mice, and 110.87 ± 31.76 in female mice (P = 0.41) in models with relative even numbers of male and female animals (7.9 ± 8.7 mice in male and 7.4 ± 6.3) (P = 0.46) (Table 4). The overall differences of drug response between male and female mice were not significant (P > 0.05) (Fig. 4). suggesting that both genders of SCID mice are appropriate for preclinical drug testing in PDOX models of pediatric brain tumors.

Table 3a.

Compounds for treatment of 6 types of PDOX models.

Tumor type	Model number	Treatment/compounds
GBM	7	XRT; ABT888; MLN8237; TMZ; Flavopiridol; VPA; SAHA
MB	2	SVV-001; TMZ; ABT888
ATRT	1	Cisplatin
EPN	1	XRT; ABT888
DIPG	2	Flavopiridol; Panobinostat
PNET	1	SVV-001
Total	14	10

Table 3b.

In Vivo treatment scheme of PDOX models.

Compounds	Name in the study	Target	Solution for compounds	Treatment scheme in PDOX model	Route of administration
Alvocidib	Flavopiridol	CDK9 kinase inhibitor	1 mg/mL in 0.2 mL DMSO add 14.8 mL PBS	5 mg/kg/day ×12 days	i.p.
Alisertib	MLN8237	Aurora A Kinase inhibitor	5 mg/mL in 10% HPBCD/1% sodium bicarbonate	30 mg/kg/day x 12 days	gavage
NA	Radiation	DNA	NA	2 Gy/day x 5 days	In located brain
Temozolomide	TMZ	Alkylate/methylate DNA	In water	50 mg/kg/day ×5 day	gavage
Panobinostat	Panobinostat	HDACi	2 mg/mL in DMSO (1.3%)/DPBS	10 mg/kg (5 d on-2 d off) x 2 cycle	i.p.
Valproic acid	VPA	HDACi	In 0.9% sodium chloride solution	600 mg/mL x 7 days (1 μl/hr),	Pump
Suberoylanilide Hydroxamic Acid	SAHA	HDACi	In PBS/DMSO (1:1)	100 mg/kg, Qd, 2 weeks	i.p.
Veliparib	ABT888	PARPi inhibitor	In 2% DMSO	10μl/g ×5 days, Qd	i.p.
Cisplatin	Cisplatin	DNA	Sterile water	5 mL/kg on days 1 and 4	i.p.
Seneca Valley Virus	SVV-001	Oncolytic virus	In PBS	20μ1*10^8vp/ul for one time	Tail vein

Table 4.

Survival time in male/female mice after treatment for 6 types of PDOX models.

Gender	Number of mouse	Mouse number per model	Survival time (days)
Male	144	7.6 ± 8.7	106.9 ± 25.7
Female	140	7.4 ± 6.3 (P = 0.46)	110.9 ± 31.8 (P = 0.41)

Fig. 4.

The impact of animal gender on drug responses in PDOX models with the preclinical drug testing. A total of 284 mice from 7 GBMs, 2 MBs, 1 ATRT, 1 EPNs, 2 DIPGs and 1 PNET models were treated 10 of standard and investigational drugs/compounds, including radiation, oncolytic virus (SVV-001), ABT888, MLN8237, TMZ, Flavopiridol, VPA, SAHA, Cisplatin and Panobinostat. The survival times of male mice bearing tumors was compared with that of female mice when the mice reached the endpoint. P > 0.05 means no difference between male and female mice.

We also analyzed a total of 316 mice from 5 GBMs, 3 MBs and 1 ATRT treated with a series of 11 standard and investigational drugs/compounds from the NIH/NCI Pediatric Preclincial Testing Consortium (PPTC) (Table S2 and Fig. S1). The overall survival times were 65.4 ± 9.3 days in male mice, and 64.5 ± 10.7 in female mice (P = 0.86) with approximately numbers of male and female animals (5.4 ± 1.1 mice in male and 5.4 ± 1.1) (P = 1) (Table S2). All the mice had no significantly difference in drug response between male and female (Fig. S1). Our data showed gender of SCID mice did not have major impact on PPTC drug response in PDOX model development, that both genders of SCID mice are appropriate for preclinical drug testing in PDOX models of pediatric brain tumors.

4. Discussion

In this study, we examined if animal gender has a significant impact on the development and the drug responsiveness of pediatric brain tumor PDOX models. Our retrospective analysis of a large cohorts of PDOX models demonstrated that the gender of SCID mice did not significantly affect tumor formation, in vivo growth nor in vivo drug responses. Our data suggested that SCID mice of both genders are appropriate for brain tumor PDOX model development and preclinical drug testing.

A fundamental assumption using human tumor xenografts for cancer research is that the xenograft tumors preserve key features of the originating patient tumors. We and many other groups have been actively engaged in the establishment and characterization of PDOX mouse models to support the understanding of tumor biology and to facilitate preclinical drug testing [14-16,21,24,45-49]. Since patient tumor tissues are very difficult to obtain, every effort should be made to increase the success of model development and to enhance the predictability of preclinical drug testing in future clinical applications. For pediatric brain tumor PDOX models, we have tried to recapitulate the microenvironment of the developing brain, which is known to critically affect the biological behavior of xenograft tumors [50,51], by using young (4–6 week old) mice and by implanting brain tumor cells into the matching locations in the mouse brains [14-16,21,22,24,45,46]. While many studies favored the use of single gender (male or female) SCID mice due to various reasons, our systematic analysis support the use of both male and female mice, particularly when they were young (4–6 week), for pediatric brain tumor studies. In addition to the fact that pediatric brain tumor is not a sex-specific or gender-predominant cancer (e.g., breast cancer and prostate cancer), young age of animals and the universally low levels of sex hormones in these mice in our study may have minimized the microenvironmental differences between male and female SCID mice and contributed to their relatively homogeneous accommodation of newly implanted human tumor cells and the subsequent responses of the xenografts toward anti-cancer treatments.

One added benefit of using both genders of SCID mice, particularly in those laboratories that breed their own SCID mice, is the increased availability of animals (as all the litters can be used). In this way, large cohorts of models can be established to support biological and preclinical drug testing with significantly reduced cost of SCID mice (50% reduction as compared with the use of single gender mouse).

As preclinical drug development is one of the primary goals of animal model development, it is also important to evaluate if there is any difference of animal gender on drug responses in vivo. For pediatric brain tumors, however, no previous studies have been published about the impact of animal gender on drug response. Recognizing the vast differences of therapeutic targets and mechanisms of action, we analyzed a large collection of anti-cancer agents to minimize or prevent our risk of inaccurate or biased conclusion caused by single drugs. Analysis of 284 mice from 14 models (7 GBM, 2 MB, 1 ATRT, 1 EPN, 2 DIPG and 1 PNET) treated a series of standard and investigational drugs/compounds, including fractionated radiation, Temozolamide (TMZ), oncolytic virus (SVV-001), ABT888, MLN8237, Flavopiridol, valproic acid, SAHA, Panobinostat and Cisplatin, did not find major differences between male and female SCID mice in our PDOX models. These data combined with the results obtained from a panel of target therapies completed with the Pediatric Preclinical Testing Consortium provided the much-needed experimental data to justify and support the use of SCIC mice of both genders in preclinical drug testing for pediatric brain tumor models.

In conclusion, our retrospective analysis of a large panel of PDOX models derived from different types of pediatric brain tumors and a series of anti-cancer drugs in vivo did not identify significant differences between male and female SCID mice on tumorigenicity, growth, sub-transplantability, and in vivo drug responses. Our data, accumulated over 15 years, support the use of both genders of SCID mice for PDOX model development and preclinical drug testing.

This study demonstrated that the gender of SCID mice dids not have major impact on animal development nor in drug responses, and SCID mice of both genders are appropriate for brain tumor PDOX model development as well as preclinical drug testing.

Abbreviations

ABT888

Veliparib

ATRTs

Atypical teratoid/rhabdoid tumors

DIPGs

Diffuse intrinsic pontine gliomas

EPNs

Ependymomas

GBMs

Glioblastomas

MBs

Medulloblastomas

PDOX

Patient derived orthotopic xenograft mouse models

PENTs

Primitive neuroectodermal tumor

SAHA

Suberoylanilide Hydroxamic Acid

SVV-001

Seneca Valley Virus

TMZ

Temozolomide

VPA

Valproic acid

XRT

Radiation