Abstract 508: Triple negative breast cancer growth and immune responses to metronomic chemotherapy regimens with varied drugs and dosages
Authors: Amanda I. Rakoski, Sarah Y. Neshat, Joshua C. Doloff
Journal: Cancer Research (2025)

Abstract Despite an increasing number of chemotherapy options, triple-negative breast cancer (TNBC) has arisen as the subtype that causes the most breast cancer mortality as it is not sensitive to drugs targeting estrogen, progesterone, and epidermal growth factor 1 receptors. The current standard of care for cancer, utilizing maximum tolerated dose with a following long break for patient recovery, has not been optimal as it causes temporary tumor regression. We propose utilizing metronomic dose scheduling where a reduced amount of drug is given on a schedule with intermittent drug-free breaks. Due to findings on the utility of metronomic chemotherapy against glioblastoma (JCD, et al. Molecular Cancer) where cyclical immune cell infiltrations have been seen, we are applying a similar 6-day schedule on aggressive TNBC mouse models to investigate if we can cause similar induction of antitumor immunity and tumor regression. For our experiments, 4T1 TNBC cells syngeneic to BALB/c mice were injected orthotopically into the right mammary fat pad. Once tumors were ∼200 mm3, treatment was initiated. Various clinically relevant chemotherapies at doses shown in the literature to affect cancer growth were dissolved in vehicles and dispensed based on mouse body weight (mg/kg). Every 6 days, drug solutions were injected intraperitoneally (IP). We additionally injected 200 μg anti-PD1 (aPD1) IP starting at day -3 and every 6 days after that for chosen drugs at 100 mg/kg. Mice were sacrificed and tissues collected after 4 rounds of chemotherapy. Comparing six chemotherapies, we found no tumor regression, but tumor stasis was instead achieved in gemcitabine (GEM) and cyclophosphamide (CPA)-treated mice. After increasing GEM up to 150 mg/kg, CPA up to 200 mg/kg, and adding aPD1 to GEM and CPA at 100 mg/kg, the tumor volume still remained static vs. GEM and CPA at 100 mg/kg alone. To investigate local immune responses, qPCR was performed on bulk tumor tissues, measuring RNA expression of gene markers for immune cells. Immune trends comparing GEM and CPA varied based on drug mechanism of action. Anti-metabolite GEM caused more expression of markers associated with the innate immune system, seen with natural killer cell marker NKp46. Alkylating agent CPA-treated tumors experienced more favorable adaptive immune cell RNA expression such as increased CD8+ T cell marker CD8a and decreased FoxP3 marker seen in Tregs. Generally, decreased immune marker gene expression was seen when the mice were given higher doses compared to 100 mg/kg, suggesting dose-limiting immune cytotoxicity. aPD1 co-treated tumors also had increased immune marker expression but not enough to change tumor stasis to regression. Ultimately, these findings showcase that metronomic chemotherapy can bring upon immune cell responses, but drug dose and activation of immune cells need to be carefully considered for potential in achieving tumor regression. Citation Format: Amanda I. Rakoski, Sarah Y. Neshat, Joshua C. Doloff. Triple negative breast cancer growth and immune responses to metronomic chemotherapy regimens with varied drugs and dosages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 508.