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

## TL;DR

Removing two proteins, stefin B and cystatin C, in a breast cancer mouse model slows tumor growth and reduces spread by changing the tumor's immune environment.

## Contribution

The study reveals that combined deficiency of stefin B and cystatin C has synergistic tumor-suppressive effects not seen with single-target inhibition.

## Key findings

- Combined deficiency of stefin B and cystatin C delays tumor onset and suppresses growth in a breast cancer model.
- Increased infiltration of M1 macrophages in tumors suggests immune reprogramming toward tumor suppression.
- Dual inhibition of these proteins uncovers new therapeutic vulnerabilities in breast cancer progression.

## Abstract

What are the main findings?
Combined deficiency of stefin B and cystatin C in the PyMT breast cancer mouse model significantly delays tumor onset, suppresses primary tumor growth, and reduces the incidence of lung metastases compared with wild-type controls.Increased intratumoral infiltration of M1-polarized macrophages is proposed to disrupt tumor-associated immunosuppressive mechanisms, thereby impairing tumor cell survival and proliferation.

Combined deficiency of stefin B and cystatin C in the PyMT breast cancer mouse model significantly delays tumor onset, suppresses primary tumor growth, and reduces the incidence of lung metastases compared with wild-type controls.

Increased intratumoral infiltration of M1-polarized macrophages is proposed to disrupt tumor-associated immunosuppressive mechanisms, thereby impairing tumor cell survival and proliferation.

What are the implications of the main findings?
Simultaneous ablation of both cysteine cathepsins inhibitors uncovers therapeutic vulnerabilities that are not apparent with single-target inhibition, highlighting the potential advantage of dual-targeting strategies.These results redefine the functional paradigm of cystatin family inhibitors in breast cancer, demonstrating a context-dependent tumor-promoting role rather than a uniformly protective function.

Simultaneous ablation of both cysteine cathepsins inhibitors uncovers therapeutic vulnerabilities that are not apparent with single-target inhibition, highlighting the potential advantage of dual-targeting strategies.

These results redefine the functional paradigm of cystatin family inhibitors in breast cancer, demonstrating a context-dependent tumor-promoting role rather than a uniformly protective function.

Background/Objectives: 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). Methods: 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. Results: 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. Conclusions: 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.