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Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures., SCOPUS: ar.j, info:eu-repo/semantics/published

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Background: FOXP1, a transcriptional regulator of lymphocyte development, is abnormally expressed in some human tumors. This study investigated FOXP1-mediated regulation of tumor infiltrating lymphocytes (TIL) in untreated primary breast cancer (BC). Methods: FOXP1 expression was analyzed in tissues from primary untreated breast tumors, BC cell lines and the METABRIC gene expression BC dataset. Cytokine and chemokine expression and lymphocyte migration in response to primary tumor supernatants (SN) was compared between FOXP1hi and FOXP1lo primary BC. Finding: FOXP1 expression was higher in estrogen receptor positive compared to negative BC. FOXP1hi tumors were significantly associated with lower TIL and fewer tertiary lymphoid structures (TLS) compared to FOXP1lo BC. Silencing FOXP1 in BC cell lines positively impacted cytokine and chemokine expression with the inverse effect associated with overexpression. CXCL9, CXCL10, CXCL11, CXCL13, CX3CL, CCL20, IL2, IL21, GZMB and IFNG expression decreased while IL10 and TGFβ increased in FOXP1hi compared to FOXP1lo primary BC. Lymphocyte migration using primary BC supernatants detected decreased mobility toward FOXP1hi supernatants. FOXP1lo BC expresses higher levels of chemokines driving TIL migration. The METABRIC gene expression dataset analysis show FOXP1 expression is associated with unfavorable BC outcomes. Interpretation: These data identify FOXP1 as an important negative regulator of immune responses in BC via its regulation of cytokine and chemokine expression. Fund: Belgian Fund for Scientific Research (FNRS 3.4513.12F) and Opération Télévie (7.4636.13F and 7.4609.15F), Fonds J.C. Heuson and Fonds Lambeau-Marteaux., SCOPUS: ar.j, info:eu-repo/semantics/published

Tumor-infiltrating B cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their roles in tumor immunity are not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2+ and triple-negative BC patients from the BIG 02-98 clinical trial (10-year median follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared with normal breast tissues, which is associated with global, CD4+, and CD8+ tumor infiltrating lymphocytes (TIL); higher tumor grades; higher proliferation; and hormone receptor negativity. All B cell differentiation stages are detectable, but significant increases in memory TIL-B are consistently present. BC with higher infiltrates are specifically characterized by germinal center TIL-B, which in turn are correlated with T follicular helper (TFH) TIL and antibody-secreting TIL-B principally located in tertiary lymphoid structures. Some TIL-B also interact directly with tumor cells. Functional analyses reveal that TIL-B are responsive to B cell receptor (BCR) stimulation ex vivo, express activation markers, and produce cytokines and Igs despite reduced expression of the antigen-presenting molecules HLA-DR and CD40. Overall, these data support the concept that ongoing humoral immune responses are generated by TIL-B and help to promote effective antitumor immunity at the tumor site., SCOPUS: ar.j, info:eu-repo/semantics/published

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even w, SCOPUS: ar.j, info:eu-repo/semantics/published

The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4(+) T cells and CD19(+) B cells and a lower mean frequency of CD8(+) T cells compare with normal tissues, increasing the CD4(+)/CD8(+) T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4(+) T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies., SCOPUS: ar.j, info:eu-repo/semantics/published

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues., SCOPUS: ar.j, SCOPUS: ar.j, info:eu-repo/semantics/published

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