Triple-negative Breast Cancer Growth and Immune Responses to Metronomic Chemotherapy Regimens with Varied Drugs and Dosages

Introduction: In the last five years, breast cancer has overtaken lung cancer as the most commonly diagnosed cancer in the world. Despite the amazing feats in breast cancer research and increasing number of chemotherapy options, triple-negative breast cancer (TNBC) has arisen as the subtype that causes the most breast cancer mortality, especially in Black women, as it is not sensitive to drugs targeting estrogen receptors (ER-), progesterone receptors (PR-), and epidermal growth factor 1 (HER1-). Therefore, we work to utilize non-hormonal chemotherapies that can continuously combat the aggressiveness of TNBC by investigating drug choice, injection schedule, and dosage.

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 relies mainly on cytotoxicity. As a result, it primarily leads to deleterious side effects and temporary regression with a retaliation of a more aggressive “immune-pruned” tumor. Therefore, 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 (Doloff, et al. Molecular Cancer, 2014) where cyclical immune cell infiltrations have been seen, we are applying a similar 6-day metronomic schedule on aggressive TNBC mouse models to investigate if we can cause similar induction of antitumor immunity and tumor regression.

Materials and

Methods: 4T1 TNBC cells syngeneic to BALB/c mice were injected orthotopically into the right inguinal mammary fat pad at 500,000 cells per 200 µl serum-free media. Mice were weighed and tumor volumes measured with a caliper twice a week until mouse study completion. Once tumors were ~200 mm3, treatment was initiated (day 0). Chemotherapy drugs were dissolved in vehicle and in amounts required for designated mg drug per kg body weight. Every 6 days starting from day 0, drug solutions were made fresh and injected intraperitoneally. Mice were sacrificed and tumors collected when the body score decreased to below a 3, tumor volume was greater than 1500 mm3, tumors continued to grow after 3 rounds of every 6d injections, or day 24 was reached. For two chemotherapy candidate regimens, we additionally injected 200 μg anti-PD1 (aPD1) intraperitoneally starting at day -3 and every 6 days after that, alternating with metronomic chemotherapy.

For qPCR, tumors were homogenized, RNA extracted by Trizol protocol, cDNA was synthesized using Thermo’s High Capacity RT kit. SYBR Green and primers were used to measure expression levels amongst generated cDNA for target genes using a QuantStudio 5. Designed primers were for genes of various innate or adaptive immune cell markers present in control vs. treated tumors. Statistical analysis for expression fold change graphs was done using Brown-Forsythe and Welch ANOVA tests on pools of RNA (N = 3-5).

Results, Conclusions, and Discussions: To identify metronomic chemotherapy capable of inducing anti-tumor immunity against TNBC, we treated BALB/c mice with orthotopic 4T1 tumors with various clinically relevant drugs at doses shown in the literature to affect breast cancer growth (Table 1). Most drugs did not negatively affect body weight except doxorubicin (DOX) at 4 mg/kg (Fig. 1A). Only gemcitabine (GEM) and cyclophosphamide (CPA) were capable of inducing tumor stasis (Fig. 1B), with 4 other compounds proving ineffective. We next investigated optimal metronomic dosing of our two lead agents (Fig. 2). Increasing GEM led to similar tumor stasis and eventual delayed growth compared to GEM 100 (Fig. 2A). We also incorporated 200 µg of checkpoint inhibitor anti-PD1 (aPD1) (every 6 days), 3 days off-set from 6-day metronomic chemotherapy, to investigate whether immune suppression was interfering with therapy; however, tumor stasis still resulted. Interestingly, adding aPD1 to CPA 100 instead increased the rate of tumor growth compared to CPA 100 alone, suggesting that such combinations are not a one-size-fits-all and must be considered carefully.
To investigate local immune responses qPCR was performed on bulk tumor samples (Fig. 3). We saw differences in immune trends, based on drug mechanism of action. Anti-metabolite GEM-treated tumors had more expression of NKp46 (NK cell marker) (Fig. 3A) while more CD8a (CD8+ T cell marker) expression (Fig. 3B) was found in alkylating agent CPA-treated groups. There was also a trend where GzmB expression decreased as dose increased for both GEM and CPA, suggesting each drug could become immunosuppressive if overdosed (Fig. 3C). Further, combined aPD1 treatment generally displayed lower gene expression for markers associated with anti-tumor response. Lastly, we confirmed the literature with both CPA and GEM that elevated metronomic dosing can decrease FoxP3 (Treg marker) expression (Fig. 3D).

Ultimately, these findings showcase that few drugs were able to achieve stasis of aggressive 4T1 TNBC tumors when used metronomically. In addition, it is important to carefully consider drug dose in addition to choice/combination. In future work, we will further investigate intratumoral immune landscapes as we believe anti-tumor immunity is being stifled by pro-tumor immune signaling, causing an overall net immune suppression.

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