Elimination of the chemotherapy resistant subpopulation of 4T1 mouse breast cancer by haploidentical NK cells cures the vast majority of mice

Abstract

Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer.

Icaritin Synergistically Enhances the Radiosensitivity of 4T1 Breast Cancer Cells

“…For this, the clonogenic assay was used to determine the extent of reproductive death caused by single treatment of ICT or radiation alone or both in combinations. Since the mouse 4T1 breast cancer cell line is one of the aggressive tumor cell lines that can grow as primary or metastatic tumor in BALB/c mice [26] and is relatively resistant to chemotherapy and radiotherapy [27], it was chosen as the targeted cells. The result (Table 1) demonstrated that when ICT was added to these highly malignant cancer cells, the IR dose required for reducing the fraction of colonies to 37% was dropped from 5.5 Gy to 4.7 Gy (at ICT 3 µM) or 3.7 Gy (at ICT 6 µM), respectively.…”

Bone marrow produces sufficient alloreactive natural killer (NK) cells in vivo to cure mice from subcutaneously and intravascularly injected 4T1 breast cancer

“…1–4, “only s.c. 4T1 tumor induction” groups). When these tumor-bearing mice were subsequently treated with the same low-dose chemo-irradiation as used in the above and previously described [1] transplantation experiments at 8 and 9 days after 4T1 tumor induction, tumor growth was permanently arrested in the vast majority of mice (Table 1, exp. nos.…”

“…injected 4T1 breast cancer is eradicated by AGM1- and NK1.1-positive cells. As only NK cells share both characteristics [58, 59] and because NK cells alone are sufficient for cure in the NK cell transfer model [1] [and this manuscript], it is apparent that the anti-tumor effect resulted from the MHC-mismatched host’s activated NK cells.…”

“…Whereas all NK cells of B6 (H-2 b ) and B6CBAF1 (H-2 b/k ) mice are alloreactive toward H-2 d target cells (i.e., “full-alloreactive”), only half of the NK cells in CB6F1 (H-2 b/d ) mice are (i.e., “half-alloreactive”). This difference has been shown to translate in faster in vivo elimination of H-2 d -positive target cells by full-alloreactive NK cells than by half-alloreactive NK cells [1, 27, 28]. Besides, CB6F1 T cells are tolerant to Balb/c tissue and are therefore not able to act as alloimmune effector cells.…”

“…Mice with 4T1 breast cancer can be cured by transfer of 5 million alloreactive NK cells after a non-myeloablative dose of total body irradiation and cyclophosphamide (“chemo-irradiation”) [1]. NK cell alloreactivity is present when tumor cells do not express the appropriate major histocompatibility (MHC) alleles for one or more inhibitory receptors of donor NK cells (i.e., “missing self” concept [2]), a situation that typically may occur when using MHC-mismatched donors.…”