Female balb cj

Female BALB/cJ and C57Bl/6 (B6) mice, six to eight weeks old, were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained under pathogen-free conditions in animal facilities and were fed autoclaved food ad libitum. Mice were housed and cared for in standard IACUC-approved animal facilities at Bloodworks Northwest and used in compliance with IACUC-approved protocol 5285-01, which adheres to the NIH Office of Laboratory Animal Welfare standards. Mice were anesthetized using isoflurane and final bleed was collected by cardiac puncture, before been cervically dislocated inside a biosafety cabinet. For organs collection, mice were then doused with 70% ethanol and opened using sterile scissors and forceps. All methods are reported in accordance with ARRIVE guidelines.

The primary TNBC xenograft mouse models were generated by injecting 1 × 10⁶ cells of 4T1-FLuc cells into the fourth mammary fat pad of the six-week-old BALB/cJ female mice (Jackson Labs, Bar Harbor, ME, USA) [52 (link),53 (link),56 (link)]. Mice were randomized into eight groups (n = 5–6) when tumor volume reached ~100 mm³ within 10–14 days post cells implantation. These models were used to analyze the possible toxicity, evaluate the dosage effect of mAb-EV-Ver-A and GC, and test the anti-TNBC efficacy of the dual-targeted therapy. Specifically, the TNBC xenograft mice were treated with 2 mg/kg-BW of GC with i.p. injection daily for the first six days. Then mice were treated with five dosages of EGFR/CD47 mAb-EV-Ver-A (i.e., 0, 0.5, 1.5, 2, and 2.5 mg/kg), 0.5 mg/kg of EGFR mAb-EV-Ver-A, 0.5 mg/kg of CD47 mAb-EV-Ver-A, and mAb-EV via i.v. injection on a Q3D × 4 schedule (i.e., three-day interval for four injections). Tumor volume was measured using an electronic caliper and calculated as “width × width × length/2”. The body weight data were collected every other day. The in vivo treatment study was ended when tumor volume reached >1000 mm³ in the control (PBS) group. At the end of the experiment, mice were euthanized and sacrificed to collect TNBC tumor and major organs for H&E staining to evaluate the possible toxicity.

This study was approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC ID: AMEND201900174/PROTO201900053, August 26, 2019) Weanling mice (Balb/cJ female) were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained on a plant protein-free diet (AIN-93M) throughout the study. Mice were used for sensitization when they turned 4–6-weeks old. Age- and gender- matched mice were used as the control mice and were kept in the same room. All animal procedures used were in accordance with Michigan State University policies.