22Rv1 (1 × 106) were suspended in 100 µl of saline with 50% rat-tail collagen and were implanted subcutaneously into the flank of 6-week-old male athymic nude mice (Envigo, Indianapolis, IN). N = 6 mice were used per a condition, based on previous subcutaneous tumor experiments. When average tumor volume reached 80 mm3, the mice were placed into four groups (IgG alone, TRC105 alone, IgG with radiation, and TRC105 with radiation) by randomization and the first dose of TRC105 or IgG was administered. Mice were treated with either IgG or TRC105 (50 µg) three times a week, unblinded. Tumor volume was recorded three times a week with digital calipers. No animals were excluded from analysis. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center.
Male athymic nude mice
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Male athymic nude mice are laboratory animals that lack a functional thymus gland, resulting in a compromised immune system. They are commonly used in medical research and drug development due to their ability to accept transplanted tissues and cells from other species without rejection.
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9 protocols using male athymic nude mice
1
Subcutaneous Tumor Xenograft Model
2
Xenograft Tumor Studies in Mice
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Male athymic nude mice were obtained from Envigo (Houston, TX, USA) at 5 weeks of age and kept under controlled conditions (12 h light and dark cycles). The animal protocol was approved by the Institutional Animal Care and Use Committee, of the Baylor & Scott and White Research Institute. Xenograft tumors were generated using the HCT116 cell line with stably knocked down TIAM1 or its scrambled controls. Cancer cells were suspended in PBS and Matrigel (Corning; 1:1 ratio) and 1 × 106 cells were subcutaneously injected into the back of each mouse. Matrigel was used to improve the attachment and differentiation of the cells. Twenty mice were used in each group, and subcutaneous tumors were monitored for 21 days following injection. In each group, ten mice were injected with 50 mg/kg of 5-FU every three days, and ten mice in the control group were injected with DMSO at the same frequency. The tumors were measured every two days. Tumor size was measured using calipers and the volume was calculated using the following formula: L × W2 × 0.5, where L represents length and W width. At 21 days post-injection, all animals were sacrificed. Tumor samples were dissected and stored in RNA-later (MilliporeSigma, St. Louis, MO, USA).
3
Aspirin Intervention in CRC Xenograft
4
Chemopreventive and Anti-tumor Evaluation
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All studies were approved by the Institutional Animal Care and Use Committee. In rat experiments, Pirc males at 5 months of age were assigned to study groups (3–4/group). Rats were then treated for 2 months with test agents, as follows: 6-SFN, 10 mg/kg body weight via daily oral gavage (p.o.); JQ1, 10 mg/kg body weight via twice weekly intraperitoneal (i.p.) injection; 6-SFN + JQ1 at the doses of the individual compounds, or vehicle. At the end of the study, colon polyps were enumerated, and tissues were bio-banked for molecular analyses, as reported [13 (link),33 (link),36 (link)].
Xenograft experiments were done as previously described [13 (link)]. Briefly, SW620 cells (5 × 106) were injected into either flank of male athymic nude mice (Envigo, Indianapolis, IN, USA). After a week, animals were randomized to treatment groups, as follows (n = 5 mice/group): BG45, 50 mg/kg body weight; dBET6, 7.5 mg/kg body weight, thrice weekly i.p. injections for 4 weeks; BG45 + dBET6, at the doses of the individual compounds, or vehicle. Tumor volumes were measured twice per week using Vernier calipers.
Xenograft experiments were done as previously described [13 (link)]. Briefly, SW620 cells (5 × 106) were injected into either flank of male athymic nude mice (Envigo, Indianapolis, IN, USA). After a week, animals were randomized to treatment groups, as follows (n = 5 mice/group): BG45, 50 mg/kg body weight; dBET6, 7.5 mg/kg body weight, thrice weekly i.p. injections for 4 weeks; BG45 + dBET6, at the doses of the individual compounds, or vehicle. Tumor volumes were measured twice per week using Vernier calipers.
5
Colon Cancer Chemopreventive Strategies
6
Evaluating Combination Therapy for Tumor Suppression
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Seven week-old male athymic nude mice (Envigo, Houston, TX) were housed under controlled conditions of light and fed ad libitum. Approximately 1 × 106 HCT116 cells were suspended in Matrigel matrix (BD Biosciences) and subcutaneously injected into mice using 27-gauge needle (n = 10 per group). Mice were randomly assigned to different treatment groups and orally gavaged with vehicle (glycerol:water, 1:1) or OPCs or curcumin (100 mg/kg body weight dissolved in vehicle) or OPCs-curcumin combination (100 mg/kg each and 50 mg/kg each) on alternative days for 15 days. Tumor size was measured each day by calipers. Tumor volume was calculated using the following formula: 1/2(length × width × width). The investigator was not blinded to the group allocation during the experiment and/or when assessing the outcome. The animal protocol was approved by the Institutional Animal Care and Use Committee, Baylor Scott & White Research Institute, Dallas, Texas and all experiments were conducted strictly in accordance to the National Institute of Health Guide for the Care and Use of Laboratory Animals (8th Edition Institute for Laboratory Animal Research).
7
Subcutaneous Tumor Implantation and Treatment
8
Xenograft Tumor Regression with Andrographis and OPCs
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Seven-week-old male athymic nude mice (Envigo, Houston, TX) were housed under controlled light conditions and were provided with food and water ad libitum. Xenograft tumors were generated by subcutaneous injection of 1 × 106 HCT116 cells. Tumor volume was calculated using the formula: (1/2) (length × width × height). Animals were randomly divided into four groups with 10 animals in each group: (1) untreated vehicle (PBS), (2) 50 mg OPC/kg body weight daily, (3) 125 mg andrographis/kg body weight every alternate day or (4) andrographis and/or OPCs together at the concentrations listed above. All treatments were injected intraperitoneally daily for 15 days, followed by euthanasia. Tumor samples were dissected, weighed and stored in RNAlater (Sigma-Aldrich) for further analysis. The animal protocol was approved by the Institutional Animal Care and Use Committee, Baylor Scott & White Research Institute, Dallas, Texas (Ethics code; A18-004, Approval date; 12/10/2018). All experiments were performed in accordance with relevant guidelines and regulations. The human equivalent dose was calculated using following formula as described previously29 (link).
9
Xenograft Model for Targeted Therapy Evaluation
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Xenograft experiments received prior approval from the Institutional Animal Care and Use Committee, and were performed on n = 5–6 animals/group7 (link). In brief, 5 × 106 SW620 cells were injected into either flank of male athymic nude mice (Envigo). After a week, animals were treated with vehicle or 7.5 mg/kg body weight of dBET6, thrice per week for 3 weeks by i.p. injection. Tumor volumes were measured twice per week using Vernier calipers, and the corresponding tumors were examined at the end of the study for molecular target modulation, as reported7 (link). In brief, xenografts were lysed using IP lysis buffer containing protease inhibitors7 (link), followed by immunoblotting of target proteins, as described above.
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