Crl 2539

In vitro safety and efficacy of AuNPs were assessed in murine healthy fibroblasts L-929 (CCL-1^TM, ATCC^®, Manassas, VA, USA), murine breast cancer 4T1 cells (CRL-2539™, ATCC^®, Manassas, VA, USA) and human breast cancer MCF-7 (HTB-22^TM, ATCC^®, Manassas, VA, USA) and MDA-MB-231 cells (HTB-26^TM, ATCC^®, Manassas, VA, USA). The MCF-7 cell line represents an estrogen receptor (ER) and progesterone receptor (PR) positive and HER2 negative cancer [61 (link)], whereas MDA-MB-231 and 4T1 cell lines represent triple-negative cancer [62 (link),63 (link)]. All cell lines were cultured in DMEM with high glucose (4500 mg/L) enriched with 10% of FBS (v/v), 100 IU/mL of penicillin and 100 µg/mL of streptomycin (henceforward, complete medium). Cells were kept in an incubator (NuAire NU-5500E, NuAire, Plymouth, MN, USA) at 37 °C and 5% CO₂ atmosphere, and every two days cell medium was changed when a confluence of 80% was reached.

4T1 mouse mammary carcinoma cells were purchased from ATCC (ATCC® CRL-2539™, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (Gibco, Montreal, Quebec, Canada) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco). The cells were harvested by brief exposure to 0.25% (w/v) trypsin-ethylenediaminetetraacetic acid, washed 3 times with Dulbecco's modified Eagle medium, and suspended in the phosphate buffered saline (PBS) at 1×10⁶ cells/mL [13 (link)].

Animal procedures were approved by the Government of Upper Bavaria. Female athymic Foxn1 nude mice (5-week old, Envigo, Germany) were injected into the mammary fat pad with 4T1 murine breast cancer cells (CRL-2539, ATCC, Manassas, VA, USA; 1 × 10⁴ cells/animal) or KPL4 human breast cancer cells^{71 (link)} (kindly provided by J. Kurebayashi, Kawasaki Medical School, Kurashiki, Japan; 2 × 10⁶ cells/animal). NOD/SCIDSHrN nude mice (5-week old, Charles River Laboratories, Germany) were injected into the mammary fat pad with MDA-MB-231 human breast cancer cells (CRM-HTB-26, ATCC; 3 × 10⁶ cells/animal). Each type of cancer cell was injected into three animals under isoflurane anaesthesia. When tumours reached a diameter of ~8 mm, the animals were anaesthetized and imaged using MSOM. This tumour size was reached within 7–10 days in 4T1-injected animals, within approximately 40 days in KPL4-injected animals, and in 30–40 days in MDA-MB-231-injected animals. At this time, a mouse carrying a 4T1 tumour was injected intravenously with 50 μl of gold nanoparticles (D12M-780–50, Nanopartz, USA) suspended in 200 μl of phosphate-buffered saline (PBS) and then imaged in vivo.

Female BALB/c mice, 6–8 weeks old (Hylasco Biotech (Charles River license, India), were used for determining the efficacy of AVA-NP-695 in vivo. 4T1 Murine Breast cancer cells (ATCC^®® CRL-2539™; 0.1 × 10⁶ cells in 1× HBSS) were implanted subcutaneously into the mammary fat pad for developing 4T1 syngeneic tumor models. The tumor-bearing animals were then randomized into 6 different treatment groups of 8 animals per group once the tumor size reached ~50–60 mm³. These mice were treated with AVA-NP-695 (1 mg/kg, 3 mg/kg, and 6 mg/kg, BID) and Olaparib (50 mg/kg) orally once daily until the end of the study. Anti-PD1 antibody (10 mg/kg) was administered IP every 3 days. Tumor growth was determined using a digital vernier caliper thrice in a week along with measurements of body weight. The efficacy of the test compound was assessed in terms of tumor growth inhibition (TGI). Percentage tumor growth inhibition (% TGI) between the control and the treated groups was calculated. At the end of the study, lungs were excised and visually observed for metastatic cancer cell colonies. Post-isolation, lungs were fixed in Bouin’s solution and surface lung nodules were counted. All lung specimens were preserved in 10% NBF for further histological evaluation of metastasis.