Background In 2020, over 42,000 women in the US are expected to die from Breast Cancer (BC). Triple Negative Breast Cancer (TNBC), a subtype defined by lack of estrogen receptor (ER), progesterone receptor (PR) and HER2 amplification, account for 15–20% of all BC. TNBC is more prevalent in pre-menopausal African-American and Hispanic women. Currently, chemotherapy is the standard of care for TNBC. Unfortunately, despise the high rate of initial response to neo-adjuvant chemotherapy, TNBC have higher rates of distant recurrence, and few (less than 30%) of the patients survive more than 5 years. Even though this subtype express high levels of PD-L1, the response to checkpoint inhibitor therapy have been modest. We hypothesized that the induction of cell death (Docetaxel) coupled with an immuno-activated milieu (locally injected adv.IL-12) would prime the tumor to respond to Anti-PD1 therapy. In this study, we investigated the effects of initially treating TNBC with a single dose of Docetaxel and adv.IL-12, followed by Anti-PD1 in syngeneic models.
Methods Syngeneic E0771 and 4T1 cell lines were injected in the mammary fat pad of C57BL/6, and Balb/c mice respectively. On day 0, mice in the Triple Combo group received a single dose (20 mg/kg) of Docetaxel and an intratumoral injection (1.25 × 109) of mAdv.IL-12 (a replication defective adenoviral vector containing mouse IL-12 cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat) (provided by Dr. Chen), followed by IP injection Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) on days 3,5,7,10,12, and 14. The other groups, received single therapy following the same procedure. On day 19, Tumor Infiltrating Lymphocytes (TILS) were isolated by Ficoll gradient and submitted for immuno-phenotyping by CyTOF analysis to the HMRI ImmunoMonitoring Core, in addition, tumor lysates were used to measure cytokine expression using Millipore Sigma’s Milliplex MAP Mouse Cytokine/Chemokine Magnetic Beads panel (cat: MCYTMAG-70K). Survival status over time, as well as tumor volume (measured every 3 days) were monitored in both models.
Results Triple combination inhibited tumor growth in the 4T1 model while significantly delaying E0771 tumor progression. Triple Combo (TC) group had significantly higher number of TILS in both models, while the phenotype and cytokine expression significantly differed. In 4T1, TC increase the infiltration of both CD8 and CD4 effector cells, while significantly decreasing neutrophils. The levels of G-CSF, Rantes were significantly upregulated in this model, while pro-tumorigenic cytokines such as IL-6, LIF, IL-1b, and anti-inflammatory cytokines such as IL-9 and IL-10 were downregulated. In E0771, only effector, and IFN-g producing CD8 levels were increased in TC group. Although TC treated animals survived an average of 18 days more than single Doc treated animals, levels of IL-6, IL-1b, LIF, KC, TNFa and VEGF levels were higher at the end of the study.
Conclusions Ad.IL-12 plus Docetaxel followed by Anti-PD1 therapy appears to only be beneficial to a specific subgroup of TNBC. We are actively studying the molecular difference between the two models used in this study, as well as investigating the clinical relevance of these markers using our extensive repertoire of PDXs in a humanized mice model.
Ethics Approval The study was approved by the Houston Methodist Research Hospital IACUC committee AUP: 0320-0023
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