Hu-gu-xiao-ji formula reprograms macrophage polarization and suppresses tumor growth to improve non-small cell lung cancer with bone metastasis via multiple signaling pathways.

[BACKGROUND] Bone metastasis represents the primary cause of mortality and a major therapeutic challenge in advanced non-small cell lung cancer (NSCLC). Hu-gu-xiao-ji (HGXJ) formula has been proved to alleviate bone-metastatic pain in our previous study while its antitumor effect and mechanism remained unclear.

[PURPOSE] This study is designed to explore the potential efficacy and underlying mechanisms of HGXJ formula against NSCLC with bone metastasis.

[METHODS] Mouse xenografts and bone-metastatic models were applied to validate the inhibitory effect of HGXJ formula on primary and bone-metastatic NSCLC in vivo. Immunological experiments were conducted to assess the immune influence of tumor and bone microenvironment or peripheral circulation under the treatment with HGXJ formula monotherapy or in combination with immune checkpoint inhibitors (ICIs). Meanwhile, transcriptome sequencing, network pharmacology analysis, and molecular biological experiments were used to investigate the underlying mechanisms of HGXJ formula in modulating tumor progression and immune microenvironment.

[RESULTS] HGXJ formula significantly suppressed the growth of primary NSCLC without observable toxicity and demonstrated efficacy against bone metastasis and its secondary dissemination, either alone or combined with ICIs. Furthermore, HGXJ formula reduced M2-type macrophages polarization and enhanced CD8T cells infiltration to remodel tumor and bone microenvironment. Mechanism investigations further confirmed the dual function and muti-pathway modulation of HGXJ formula on directly inhibiting tumor via MAPK cascade and diminishing macrophages M2 polarization via STAT3/6 pathway, thus playing the antitumor role on bone-metastatic NSCLC.

[CONCLUSION] These findings revealed the multifaceted therapeutic effects of HGXJ formula and provided mechanistic evidence supporting its potential as a candidate therapeutic option for NSCLC patients with bone metastasis.