Stefin B and Cystatin C Deficiency Suppresses Tumor Growth and Alters Tumor Microenvironment in a Breast Cancer Model

<b>Background/Objectives</b>: Cysteine cathepsins and their endogenous inhibitors have been shown to possess context-dependent functions in cancer progression, including the regulation of tumor metabolic pathways. Stefin B and cystatin C, intracellular and extracellular protease inhibitors, respectively, can modulate tumor biology through protease-dependent and protease-independent mechanisms. This study investigated their combined functions and potential roles as tumor promoters in breast cancer in a spontaneous breast cancer mouse model (PyMT mice). <b>Methods</b>: We generated PyMT transgenic mice lacking both stefin B and cystatin C (double-knockout, DKO) and compared their tumor growth kinetics, proliferation, apoptosis, and metastatic burden with those of wild-type control mice. Immunohistochemistry was performed to characterize tumor macrophage infiltration and polarization. <b>Results</b>: DKO mice demonstrated delayed tumor onset, significantly slower tumor growth, reduced proliferation, increased apoptosis, and fewer lung metastases compared to wild-type controls. Immunohistochemistry revealed enhanced macrophage infiltration of the tumors, accompanied by a pronounced shift toward antitumorigenic M1 (CD86⁺) polarization, while M2 (CD206⁺) populations remained unchanged, indicating an immunological reprogramming of the tumor microenvironment toward a pro-inflammatory, tumor-suppressive state. <b>Conclusions</b>: Our results demonstrated a potential function of stefin B and cystatin C as tumor promoters in breast cancer through complementary mechanisms. Simultaneous depletion of both inhibitors revealed synergistic effects and remodeled the immune microenvironment to favor tumor suppression. These results suggest previously unknown roles for stefin B and cystatin C in tumor development and progression, which encourage further investigation of the cancer metabolic mechanisms underlying tumor behavior and their dynamic interplay with the microenvironment.