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STAT5 Mediated Antitumor Functions in Breast Cancer

T35 Supergroup

August 21 2024

Ellis Raduenz

Schwertfeger Lab

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Introduction and Background

Jen Tuokkola & Olivia Kuckhahn, ©Biorender

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Overview of the breast cancer immune environment

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©Biorender

Tumor-associated macrophages (TAMs) can suppress or support tumor progression in a context-dependent manner

Macrophages, particularly tumor-associated macrophages (TAMs), are central to the immune landscape of cancer due to their ability to exhibit dual roles. On one hand, TAMs can adopt an M2 or pro-tumorigenic phenotype, where they secrete factors like IL-10 and TGF-β that suppress antitumor immune responses and facilitate tumor growth and metastasis. Alternatively you can think of these chemokines secreted by this M2 macrophage inhibiting the immune system leading to tumor growth/metastasis. This is often seen in the later stages of tumor development. On the other hand, TAMs can also exhibit an M1 phenotype, promoting antitumor immunity by producing pro-inflammatory cytokines and activating cytotoxic T-cells. Again, alternatively you can think of the chemokines secreted by the M1 macrophage promoting immunity which is good for cancer prognosis. The balance between these phenotypes is influenced by signals from the tumor microenvironment, making TAMs a crucial target for cancer immunotherapy.

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JAK2/STAT5 Signaling Pathway

Olivia Kuckhahn, ©BioRender

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nucleus

Extracellular Space

Cytosol

Cytokine Ligand

Jesser et. al.

PMID: 34743736

1, Ligand activates tyrosine kinase receptor

2, 3, 4 Activated JAK phosphorylate Tyr residues and recruit STAT proteins

5, Activated STAT dimerizes

6, STAT translocation to the nucleus where it regulates transcription

The STAT5 pathway plays a crucial role in immune regulation, particularly in macrophage function. STAT5 is activated by cytokine signaling, leading to their phosphorylation and movement into the nucleus. Here, they influence gene expression tied to cell survival, proliferation, and immune responses. In TAMs, the JAK2/STAT5 signaling pathway is activated by tumor cell-secreted factors such as granulocyte-macrophage colony stimulating factor (GM-CSF). In TAMs, STAT5 activation can determine whether cells adopt this pro-inflammatory (M1) or anti-inflammatory (M2) role. This pathway is key to maintaining a balanced immune environment, and its dysregulation can contribute to the suppression of immune responses in tumors, aiding cancer progression.

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Role of STAT5 in Breast Cancer

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Jesser et. al. PMID: 34743736

Aditi Bapat, Ph.D., unpublished

Olivia Kuckhahn, ©BioRender

STAT5 is increasingly recognized for its role in the tumor microenvironment (TME) of breast cancer. Specifically, in particularly aggressive forms like triple-negative breast cancer (TNBC). STAT5 signaling modulates the immune landscape driving chemokine production within macrophages, overall influencing the immune landscape. As we’ve seen before, when STAT5 is active, it may promote a pro-tumorigenic environment by encouraging macrophages to adopt an M2 phenotype, which supports tumor growth and suppresses the body’s anti-tumor immune responses. On the other hand, inhibiting STAT5 in these cells could reduce their support of tumor progression, making the immune environment more hostile to cancer cells. The LysMCre+ mouse model, with myeloid-specific STAT5 deletion, is used to study these differing phenotypes on the TME.

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Role of STAT5 in Breast Cancer

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Jesser et. al. PMID: 34743736

Aditi Bapat, Ph.D., unpublished

Olivia Kuckhahn, ©BioRender

Deletion of STAT5 in myeloid cells increases metastasis in mouse models of breast cancer, suggesting an anti-tumor function of STAT5 in macrophages.

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Hypothesis & Rationale

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©Biorender

Impact of STAT5 deletion in TAMs on tumor immune suppression

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Methods, Results, & Future Directions

Immunofluorescent staining of CD8+ T-cells in primary and metastatic tumor sections

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A. Image overlay in primary tumor core with fluorescent blue DAPI counterstain

B. Fluorescent green CD8+ staining in primary tumor.

C. Image overlay in lung metastasis tissue with fluorescent blue DAPI counterstain.

D. Fluorescent green CD8+ staining in lung met tissue periphery.

All images at 40x magnification.

40x magnification Keyence BZ-X series microscope

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CD103+CD8+ T cells localize to epithelial cell regions in breast tumors and non-cancerous breast tissues

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Egleston et. al. PMID: 31465302

Lai et. al. PMID: 33479027

CD103+ TRMs in cutaneous SCC are situated predominantly in peritumoral areas

Recent studies have highlighted the critical role of spatial localization of CD8+ T-cells within tumor microenvironments, particularly within cancer islands, in predicting relapse-free survival (RFS) in breast cancer patients. Egleston’s work shows that the effectiveness of CD8+ T cells in controlling breast cancer is highly dependent on their localization within these cancer islands rather than just their presence in the peritumoral / stroma area. Localization into cancer islands within tumors is mediated by expression of the integrin CD103, which is a marker for tissue-resident memory T cells (TRMs). Thus, this research underscores the importance of CD8+ tissue-resident memory T cells (TRMs) that localize in proximity to cancer cells as being better positioned to exert anti-tumor effects, leading to improved outcomes.

In contrast, on the right, we can see that in a different immunologic disease model, cutaneous SCC, CD8+ cells are localized to the peritumoral / stroma area. I mainly want you all to appreciate the quality of their staining!

My research aims this summer were to similarly map the spatial distribution of CD8+ T-cells in both primary and metastatic tumor samples, to better understand their role in antitumor immunity.

Non-cancerous breast tissues = NCBTs

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Immunohistochemical staining of Foxp3 in primary breast tumor sections

A. 1:100 dilution of of R&D Systems primary antibody.

B. 1:100 dilution of Invitrogen primary antibody.

20x magnification Keyence BZ-X series microscope

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Examples of main tumor CD8 and FOXP3 expression as assessed with immunohistochemistry in breast cancer.

Lee et. al. PMID: 28621227

CD8+ T lymphocytes in breast carcinoma

Foxp3+ lymphocytes in breast carcinoma

200x magnification

As we move forward, my primary focus will be on refining a Foxp3 IHC staining protocol to enhance both specificity and sensitivity, crucial for accurately identifying regulatory T-cells (Tregs) in our primary and metastatic tissue samples.

I discussed my IHC optimization both in FFPE and frozen sections, in my poster presentation. The goal is to ensure robust and reproducible results across different tissue preparations and to conduct IF staining for Foxp3 alongside other key immune cell markers to achieve a more comprehensive understanding of the tumor microenvironment.

Other immune cell markers of interest:

CD68: A marker for macrophages, particularly tumor-associated macrophages (TAMs), which can either promote or inhibit tumor growth depending on their polarization (M1 vs. M2).
CD4: A marker for helper T cells, including Tregs, which play a role in immune suppression within the tumor microenvironment.
Granzyme B: An enzyme found in cytotoxic T cells and natural killer (NK) cells, associated with the induction of apoptosis in target cells, and a key effector in anti-tumor immune responses.
PD-1/PD-L1: Immune checkpoint markers that are important in the regulation of immune responses. PD-1 is expressed on T cells, while PD-L1 is expressed on tumor cells and immune cells within the tumor microenvironment. Their interaction can inhibit T cell function, leading to immune evasion by the tumor.

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Acknowledgements

Schwertfeger Lab

Alexis Effstrum

Emma Nelson

Lyndsay Reese

Annisa Rumanhorbo

Jennifer Tuokkola

Olivia Kuckhahn

Yuyu He, MD

Kaylee Schwertfeger, Ph.D.

Albert Barrios

Mariajose (MJ) Franco

Department of Medicine-Rheumatic and Autoimmune Diseases

Daniel Mueller, M.D.

Drew Keup