The online version of this article (https://doi.org/10.1186/s13058-018-1037-4) contains supplementary material, which is available to authorized users.
Roberto R. Rosato and Daniel Dávila-González contributed equally to this work.
Breast cancer has been considered not highly immunogenic, and few patients benefit from current immunotherapies. However, new strategies are aimed at changing this paradigm. In the present study, we examined the in vivo activity of a humanized anti-programmed cell death protein 1 (anti-PD-1) antibody against triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tumor models.
To circumvent some of the limitations posed by the lack of appropriate animal models in preclinical studies of immunotherapies, partially human leukocyte antigen-matched TNBC PDX tumor lines from our collection, as well as human melanoma cell lines, were engrafted in humanized nonobese diabetic/severe combined immunodeficiency IL2Rγnull (hNSG) mice obtained by intravenous injection of CD34+ hematopoietic stem cells into nonlethally irradiated 3–4-week-old mice. After both PDXs and melanoma cell xenografts reached ~ 150–200 mm3, animals were treated with humanized anti-PD-1 antibody or anti-CTLA-4 and evaluated for tumor growth, survival, and potential mechanism of action.
Human CD45+, CD20+, CD3+, CD8+, CD56+, CD68+, and CD33+ cells were readily identified in blood, spleen, and bone marrow collected from hNSG, as well as human cytokines in blood and engrafted tumors. Engraftment of TNBC PDXs in hNSG was high (~ 85%), although they grew at a slightly slower pace and conserved their ability to generate lung metastasis. Human CD45+ cells were detectable in hNSG-harbored PDXs, and consistent with clinical observations, anti-PD-1 antibody therapy resulted in both a significant reduction in tumor growth and increased survival in some of the hNSG PDX tumor lines, whereas no such effects were observed in the corresponding non-hNSG models.
This study provides evidence associated with anti-PD-1 immunotherapy against TNBC tumors supporting the use of TNBC PDXs in humanized mice as a model to overcome some of the technical difficulties associated with the preclinical investigation of immune-based therapies.
Additional file 1: Representative figure showing the results of flow cytometric analysis of human cells collected from blood of nonhumanized and humanized NSG mice after 8, 16, and 22 weeks of intravenous injection of human CD34+ hematopoietic stem cells (HSCs). Procedures and antibodies used in these studies are described in the Methods section. (PPTX 722 kb)
Additional file 2: Table S1 Analysis of HLA type in PDX BCM-2147/-4913 and CD34+ HSCs. HLA typing was performed by using PCR-SSO DNA-based procedures. The serological phenotype is an interpretation based on molecular typing data. ND Not determined. Table S2 Gene expression analysis (RNA-Seq) comparing MC1, BCM-2147, and BCM-4913 PDXs growing in nonhumanized vs. humanized NSG mice. Differentially expressed genes (DEGs) were selected by edge R-based p value and fold change (FC). Supplemental Methods. (DOCX 22 kb)13058_2018_1037_MOESM2_ESM.docx
Additional file 3: IHC analysis of human CD4-, CD3-, CD8-, CD20-, CD68-, CD4-, and CD8-positive cells present in BCM-2147, MC1, and BCM-4913 tumor xenograft lung micrometastases. Representative IHC images of obtained using preparations of tumor samples grown in humanized NSG mice; 4× and 20× magnifications are shown counterstained with hematoxylin. (PPTX 3007 kb)13058_2018_1037_MOESM3_ESM.pptx
Additional file 4: Effects of the anti-CTLA-4 immune checkpoint inhibitor antibody ipilimumab against MC1 PDXs implanted in hNSG mice. Once tumors reached ~ 150 mm3, animals were treated weekly with 10 mg/kg intravenous injections for up to 3 weeks; tumor volumes were evaluated twice weekly. The values represent the mean ± SEM (n = 8). (PPTX 50 kb)13058_2018_1037_MOESM4_ESM.pptx
Additional file 5: a Evaluation of the percentages of human CD45+ TILs present in MC1 PDX tumors engrafted in hNSG mice and collected from animals treated with either vehicle control or anti-PD-1 antibody. The values represent the mean ± SEM (n = 8). b Schematic representation of the method used to determine the cytotoxic activity of TILs by measuring the levels of the lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon TIL-induced tumor cell lysis. TILs were isolated from MC1 PDX tumors engrafted in hNSG mice and treated with either vehicle or anti-PD1 antibody that were cocultured with disaggregated MC1 tumor cells obtained from the corresponding PDX grown in nonhumanized NSG mice. (PPTX 131 kb)13058_2018_1037_MOESM5_ESM.pptx
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- Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models
Roberto R. Rosato
Dong Soon Choi
Anthony J. Kozielski
Jenny C. Chang
- BioMed Central
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