An improved syngeneic orthotopic murine model of human breast cancer progression

Abstract

Breast cancer drug development costs nearly $610 million and 37 months in preclinical mouse model trials with minimal success rates. Despite these inefficiencies, there are still no consensus breast cancer preclinical models. Murine mammary adenocarcinoma 4T1-luc2 cells were implanted subcutaneous (SQ) or orthotopically percutaneous (OP) injection in the area of the nipple, or surgically into the chest 2nd mammary fat pad under direct vision (ODV) in Balb/c immunocompetent mice. Tumor progression was followed by in vivo bioluminescence and direct measurements, pathology and survival determined, and tumor gene expression analyzed by genome-wide microarrays. ODV produced less variable-sized tumors and was a reliable method of implantation. ODV implantation into the chest 2nd mammary pad rather than into the abdominal 4th mammary pad, the most common implantation site, better mimicked human breast cancer progression pattern, which correlated with bioluminescent tumor burden and survival. Compared to SQ, ODV produced tumors that differentially expressed genes whose interaction networks are of importance in cancer research. qPCR validation of 10 specific target genes of interest in ongoing clinical trials demonstrated significant differences in expression. ODV implantation into the chest 2nd mammary pad provides the most reliable model that mimics human breast cancer compared from subcutaneous implantation that produces tumors with different genome expression profiles of clinical significance. Increased understanding of the limitations of the different preclinical models in use will help guide new investigations and may improve the efficiency of breast cancer drug development .

Figure 1. ODV produced greater cancer growth with less variability than OP or SQ

Four days after implantation of 1×10⁵ cells by the respective methods in 4 separate locations in 5 mice in each group, bioluminescence was quantified, the black line represents the mean for each group of N=20 (*p<0.02, **p<0.001).

Figure 2. Pathological analyses demonstrate possible cause of variabilities in OP or SQ

(A) H&E pathology demonstrated implantation along the needle tract (black arrow demonstrates skin implantation) and in the mammary gland (white arrow) after OP method. (B) ODV produced larger tumors than SQ (**p<0.001; N=8). H&E staining showed capsule formation around SQ but not ODV tumors. (C) Kaplan-Meier analysis demonstrated ODV produced shorter survival than Sq (p<0.001).

Figure 3. ODV implantation into the chest 2^nd mammary pad better mimic human breast cancer progression than into the abdomen 4^th mammary pad

4T1-luc2 cells were implanted into either abdomen 4^th mammary pad (Abdomen; closed bar) or chest 2^nd mammary pad (Chest; open bar), and ex vivo IVIS imaging was obtained 28 days after implantation. Upper panel demonstrate the percentage of total photon counts of the metastatic lesions that had tumor in the respective organs, and their representative images are demonstrated below. Vast majority of the metastases from abdomen ODV was peritoneal carcinomatosis, whereas that of chest ODV was distributed among the distant organs. N=5.

Figure 4. Less fluid is advantageous for ODV, and it is not difficult to learn

100μL ODV injection of isosulfan blue spilled out of the mammary gland (A), while 10μL ODV stayed in the mammary gland (B). Implantation of the same number of cells, 100μL versus 10μL, cause greater tissue disruption, (C) versus (D), and less cancer growth (E). ODV required approximately 3 minutes longer to perform than other methods (F) and it only required 5 attempts for a researcher with no animal experience (closed diamond) to be proficient in this technique (G).

Figure 5. ODV versus SQ: site of implantation produced tumors with different gene expression profiles

(A) Unsupervised cluster analysis using centered correlation and average linkage demonstrated that 10350 genes were commonly different between Cells vs ODV and Cells vs SQ, which implicate the difference in gene expression profile between Cells and tumor in vivo is larger than ODV vs SQ. In total 700 genes were differentially expressed between ODV and SQ tumors. (B) Supervised cluster analysis demonstrated the differential expression of 206 significant probe sets (3.1% of entire transcriptome) by greater than 1.5 fold between ODV and Sq tumors, N=8. (C) PubMed search of these 206 significant probe sets demonstrated that 75% are known targets of therapy, cancer biomarkers, or cancer antigens, with 25% of unknown clinical significance.

Figure 6. Significant Gene Interaction Networks

The gene interaction networks with a significance p≤1×10⁻³⁰.

Figure 7. ODV and SQ Differentially Expressed Genes Important for Cancer Research and Drug Development

With a significance of p<0.0001, by genome-wide microarray and qPCR validation, there was a difference in the expression of Wnt5a (A), Cxcl14 (B), Pdgfa (C), S100a16 (D), Twist2 (E), Epha3 (F), Cd59a (G), Hsp110 (H), Hdac1 (I) and Notch4(J), but not Hdac2 (I) or Notch1 (J).