Objective:

2

To investigate the

in vivo

immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC).

Methods:

2

Murine-derived triple-negative mammary carcinoma cell line

4T1 cells

was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (

n

=11 in ench): naïve

control

LD 4HF (low dose 4HF)

HD 4HF (high dose 4HF)

LD 4HF + D (low dose and doxorubicin)

and D (doxorubicin). Apart from the naïve group

each mouse received subcutaneous inoculation with 5×10

5

4T1 cells resuspended in 100 μL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation

tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2

630 mg/kg 4HF

respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment

mice organs were harvested to analyze immunological activities via immunophenotyping

gene and multiplex analysis

histological staining

and gut microbiota analysis.

Results:

2

Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (

P

<

0.05)

altered the hypoxia and overall immune lymphocyte landscape

and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover

immunosuppressive genes

cytokines

and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (

P

<

0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (

P

<

0.05).

Conclusion:

2

The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement.