Generation of tumor grafts for feline mammary carcinoma
We orthotopically transplanted 11 fresh primary FMT samples into mammary fat pads of NSG mice. Tumors grew from 9 out of 11 samples, and we successfully maintained 4 tumor lines through multiple rounds of serial transplantation (Table 3). Tumors reached approximately 500–800mm3 volumes prior to collection. Second passage tumors showed similar overall growth rate to the initial founders with a >90% rate of successful engraftment and tumor growth (41/45). After histological review of all successful cases, we found that the resultant tumor grew from PDX models preserved the histopathology from the original patient (Fig. 1A-1B, supple. fig. 1) better than the tumor growth from primary FMT-1807 cells transplantation (Fig. 1C). One tumor graft was estrogen receptor positive, one was positive for HER2 and two were ER, PR and HER2 negative, according to immunohistochemical analysis (Table 3).
We performed microsatellite STR analyses to determine whether the established PDX model retained the identical pattern of the original tumors. We performed PCR using STR markers for following the chromosomal exchange between tumor tissues from patients and from mice that occurred in established PDX models. As a result, the expression levels of STR markers were observed at the same chromosome loci in parental tumor tissues from patients, first-generation PDX model and fifth-generation PDX model (Fig. 2). These results showed that the patient and tumor xenografted mouse models preserved the same STR profiles until at least fifth-generation.
No lymphomatous transformation or lymphocytic dominant pattern was found in tumor tissues obtained from mice in tumors stained for anti-CD20 (Fig. 3A) or anti-CD3 (Fig. 3B) antibodies.
No lymphomatous transformation or lymphocytic dominant pattern was found in tumor tissues obtained from mice in tumors stained for anti-CD20 (Fig. 3A) or anti-CD3 (Fig. 3B) antibodies.
Tumor grafts emulate metastasis seen in PDX mice
Tumor metastases in these PDX lines were identified in the lung, liver, spleen, kidneys, and lymph nodes and were examined at the time of necropsy. We detected spontaneous metastasis in the axillary node, lungs, and livers of mice with tumor grafts. Tumor metastases were detected grossly, by H&E staining and immunohistochemical staining. All of the metastatic tumors were confirmed as carcinomas (Table 1, Fig. 4). Metastasis frequencies of different passages varied from 33.3% (2/6) to 100% (6/6) in FMT1807-PDX.
Immunohistochemical characteristics of PDX tumors
We analyzed the major molecular characteristics (ER, PR, HER2, pan cytokeratin, β-catenin and vimentin) of original tumors and PDX grafts following five passages in mice. The expressions of these molecular markers revealed similarities between the xenograft tumors and the patients' tumor samples showed that the PDX model retained not only the histology but also the expression of cytological characteristics were preserved (Fig. 5).
PDX tumor growth progression in mice
Tumor growth parameters of each FMT-PDX are shown in Fig. 6. To determine estrogen dependence of ER+ tumors, mice with FMT1702 tumors were subcutaneously implanted with or without estrogen pellets after oophorectomy. The FMT1702-PDX was not estrogen dependent, but tumor growth was stimulated by estrogen.
Previous studies have shown human and canine mammary tumor cells were capable of reproducing tumors in both male mice and female mice [8]. FMT1806-PDX and FMT1807-PDX grafts were implanted into female and male NSG mice to observe if there were differences in tumor growth parameters (Table 4). FMT1806-PDX and FMT1807-PDX grafts showed no statistically significant differences in terms of the frequency of tumor appearance or the time in which a tumor volume of 500 mm3 was reached. The frequency of mice that developed metastasis differed between males and females for both FMT1806-PDX and FMT1807-PDX. The percentage of metastasis found in males was reduced in contrast to females.