A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials

Abstract

In order to develop a practical model of breast cancer, with in vitro and syngeneic, immune-intact, in vivo growth capacity, we established a primary cell line derived from a mammary carcinoma in the transgenic FVB/N-Tg(MMTV-ErbB2*)NDL2-5Mul mouse, referred to as "NDL^UCD". The cell line is adapted to standard cell culture and can be transplanted into syngeneic FVB/N mice. The line maintains a stable phenotype over multiple in vitro passages and rounds of in vivo transplantation. NDL^UCD tumors in FVB/N mice exhibit high expression of ErbB2 and ErbB3 and signaling molecules downstream of ErbB2. The syngeneic transplant tumors elicit an immune reaction in the adjacent stroma, detected and characterized using histology, immunophenotyping, and gene expression. NDL^UCD cells also express PD-L1 in vivo and in vitro, and in vivo transplants are reactive to anti-immune checkpoint therapy with responses conducive to immunotherapy studies. This new NDL^UCD cell line model is a practical alternative to the more commonly used 4T1 cells, and our previously described FVB/N-Tg(MMTV-PyVT)634Mul derived Met-1^fvb2 and FVB/NTg(MMTV-PyVT^Y315F/Y322F) derived DB-7^fvb2 cell lines. The NDL^UCD cells have, so far, remained genetically and phenotypically stable over many generations, with consistent and reproducible results in immune intact preclinical cohorts.

Keywords: Breast cancer; Erbb2; Immunotherapy; Mouse model; PD-L1; Syngeneic.

Figure 1:

In vitro and in vivo growth and phenotype of NDL^UCD. a-b) Growth attributes of the NDL^UCD cell line, a) In vitro growth rate in 2D culture. b) In vivo growth rate of transplanted NDL^UCD tumor (this data has been previously published [4]). c-g) Immunohistochemistry of the NDL^UCD transplant tumors; c) ER, d) PR, e) H&E, f) ERBB2, g) KI67. Arrows indicate normal mammary glands. h) In vitro 2D culture showing cobblestone epithelial phenotype. i-k) Lung metastasis, example of a lung showing high multiplicity of NDL^UCD tumor outgrowth after tail-vein injection. i) Whole-mount photograph of a lung lobe with multiple NDL^UCD outgrowths. j) high tumor multiplicity, arrow indicates tumor shown at a higher magnification in k). k) The lung outgrowths show characteristic NDL^UCD phenotype.

Figure 2:

Expression of ErbB pathway molecules in the NDL^UCD model. a) The ErbB pathway as presented in KEGG pathways [20] colored, based on the level of expression of the indicated molecule in the RNA-Seq analysis of NDL^UCD transplant tumors (n=3). b) Expression values in the RNASeq analysis used for color coding in the “a” panel. c) Comparison of expression of ErbB pathway genes in three transplantable mouse models: NDL^UCD, SSM2^UCD, Met-1^fvb2 (Gene expression profiling was performed with Affymetrix Mouse Gene 1.0 ST Arrays (n=6/each model)). Log2 transformed values were utilized for hierarchical clustering and the results are visualized with a heat map. d) Comparison of expression of main tumor suppressor genes in the NDL^UCD, SSM2^UCD, Met-1^fvb2 models. e) Western blot of ErbB2 pathway proteins from cell cultures of three transplantable mouse models (NDL^UCD, SSM2^UCD,Met-1^fvb2) (MWM: Molecular Weight Marker).

Figure 3:

Immune cell distribution in syngeneic transplant of NDL^UCD. a-f) Representative images of immunohistochemistry on an NDL^UCD transplant tumor showing the same region: a) CD8A, b) CD4, c) F4/80, d) FOXP3, e) B220, f) H&E. g-k): Immune cell marker positive cells in the tumor adjacent stroma and in the peripheral tumor of transplanted NDL^UCD tumors and primary tumors from the GEMM. n=6 transplant tumors from 3 animals (Tx) n=3 primary tumors from 3 animals (1°). * q<0.05, **q<0.01, ***q<0.001, ****q<0.0001

Figure 4:

Illustration of heterogeneity in immune cell infiltration. a-e) Distribution of immune cells in the peripheral tumor and the tumor adjacent stroma; a) CD4, b) CD8A, c) F4/80, d) B220, e) FOXP3. f) The analyzed area shown in the tissue overview, stromal area borders shown in red, peripheral tumor area borders shown in black. g) Example of an area of high CD8 positive cell density in the peripheral tumor and in the tumor adjacent stroma. h) Example of an area with low CD8 positive cell density in the peripheral tumor and in the tumor adjacent stroma from the same tumor as g).

Figure 5:

PD-1 and PD-L1 expression in the NDL^UCD model. a) b) PD-1, CD3, ECAD expression stained with multiplex IHC. c) d) PD-1 expressing immune cells. The arrows indicate PD-1 positive cells. e) Positive PD-L1 staining, with typical zonal distribution of staining intensity. f) PD-L1 IHC on cell culture. g) PD-L1 western blot for cultured NDL^UCD cells.

Figure 6:

Immune reaction-associated gene expression in two mouse models. Gene expression profiling of NDL^UCD and Met-1^fvb2 transplanted tumors was performed using Affymetrix Gene 1.0 ST Arrays. Samples were clustered using a homologous gene list based on identification of immune infiltration from gene expression data in humans [40]. Log2 transformed and mean-centered values are illustrated.