NK cell-triggered CCL5/IFN?-CXCL9/10 axis underlies the clinical efficacy of HER2-targeted antibodies in primary HER2 positive breast cancer [RNA-seq]

Tumor-infiltrating (TI)-NK cell numbers predict better than TIL score the efficacy of HER2-targeted antibodies in primary breast cancer patients. To understand the mechanism/s underlying this association, biological processes enriched in NK cell-infiltrated as compared to NK cell-desert HER2-positive breast tumors paired by TIL score were leveraged from transcriptomic data. NK cell-infiltrated tumors were enriched in transcripts regulated by interferons and NF-kB. Among them, levels of CCL5/IFNG-CXCL9/10 positively correlated with the number of TI-NK cells in the original biopsy. Indeed, coordinated expression of CCL5/IFNG-CXCL9/10 transcripts was also evidenced in tumor biopsies from a phase II clinical trial where IFNG levels associated to the achievement of pathological complete response to anti-HER2 antibody treatment (OR 96.3, p=0.01) and correlated with their TI-NK cell score. In in vitro models, anti-HER2 antibody-dependent NK cell activation led to the secretion of CCL5/IFN-? and the subsequent production of IFN-?-dependent CXCL9/10 by bystander breast cancer cells. Ex vivo treatment of breast tumor-derived multicellular cultures with trastuzumab induced the activation of CD16+ TI-NK cells and their conversion into a CD16-CD103+ subpopulation, both endowed with CCL5 and IFN-? producing potential. CD16+ and CD16-CD103+ TI-NK cell subpopulations shared the expression of EOMES, TBX21 and several KIR genes, indicating their lineage relationship; and their proportions positively correlated with total NK cell, CD8+ and tissue-resident T cell frequencies, immune cell subsets with anti-tumor potential. Remarkably, the coordinated induction of CCL5/IFNG-CXCL9/CXCL10 expression, concomitant to the conversion of CD16+ into CD16+/-CD103+ tumor-infiltrating NK cells, was recapitulated in a humanized HER2+ breast cancer in vivo model treated with a combination of anti-HER2 antibodies and in vitro expanded human NK cells, paralleling tumor growth control. Finally, patients achieving good clinical responses to anti-HER2 antibody-based neoadjuvant treatment showed an early and coordinated increase in sera CCL5 and CXCL9/CXCL10 levels which positively correlated with TI-NK cell numbers in the corresponding diagnosis biopsy. Overall, our data point to NK cells as regulators of the tumor microenvironment through the early secretion of CCL5/IFN-? resulting in the production of CXCL9/10 and the recruitment/differentiation of immune infiltrates with anti-tumor potential, ultimately contributing to anti-HER2 antibody clinical efficacy. Paired samples of peripheral blood and treatment-naive breast tumor specimens were obtained from 3 breast cancer patients, blind to their clinicopathological features. Multicellular suspensions from tumors were obtained as described above and stained with directly labelled antibody cocktail including a-CD45-AF700 (clone 2D1), a-CD3-PerCP (clone SK7), a-CD56-APC (clone CMSSB), a-CD16-efluor780 (clone CB16), a-CD103-FITC (Clone B-Ly7) and DAPI as a viability die. Distinct tumor-infiltrating NK cell subsets were sorted based on the expression of CD16 and CD103 (CD16+; CD16-CD103+; CD16-CD103-) from the CD56+ CD3 gate in CD45+ DAPI- lymphocytes. Peripheral blood NK cells were sorted from PBMC based on their CD56bright CD16- and CD56dim CD16+ expression profile. Cell sorting was performed in a Influx sorter ( BD, YYY) at the Flow Cytometry Facility, PRBB, Barcelona.