Scientists report on the generation of mouse models of human breast cancer in which the genetic and metastatic characteristics of the grafted tumors mirror those of the cancer in the patient. Researchers at the University of Utah’s Huntsman Cancer Institute Direct say direct orthotopic transplantation of fresh human tumor tissue into immunocompromised mice reflect the tumor pathology, growth, and metastasis in the human patient from which the sample was derived.
The results, published in Nature Medicine, also showed that the ability of a tumor from an individual with breast cancer to grow as a graft is a key indicator of shorter survival time, even for patients with no sign of metastases. Alana L. Welm, M.D., and colleagues, claim their approach could pave the way to the development of models that will help predict the progression and outcome of breast cnacer in individual patients. They report their work in a paper titled “Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.”
The development of new treatments for breast cancer has been hampered by the lack of in vivo models in which the human tumor continues to behave exactly as it would in a human patient, the researchers note. Implanted breast cancer cell lines have been informative, but only partly recapitulate the genetic features and metastatic potential of tumors in individuals with breast cancer, and this doesn’t help scientists predict how drugs will perform in a clinical setting.
Transplantation of tumor tissue into immune-deficient mice is more informative in terms of predicting drug responses, but, as the Huntsman team points out, tumor graft strategies for hormone-driven cancers such as breast or prostate cancer have had limited success. “Although the majority (about 70%) of newly diagnosed breast cancers are positive for ER, this tumor type is underrepresented in mouse models because of loss of ER expression or lack of estrogen dependence.” Moreover, they note, there is a similar lack of models that show spontaneously, clinically relevant metastasis, which is the primary cause of death from breast cancer.
The team’s aim was thus to generate an animal model of breast cancer in which tumor tissue taken directly from a patient would retain the critical characteristics of the original tumor specimen, including metastasis.
To this end they transplanted 49 fresh primary tumors or metastatic breast cancer cell samples into cleared mammary fat pads of female nonobese diabetic severe combined immunodeficiency (NOD-SCID) mice. The tissue was obtained immediately following surgery or fluid drainage from 42 different individuals. Five tumor grafts were estrogen receptor and progesterone receptor positive (ER+PR+), seven were ER and PR negative (ER−PR−), and five were positive for HER2 (HER2+). Four of the grafts were from primary tumors, and eight were from metastatic effusions.
Tumors grew from 18 out of 49 samples (37%), and the researchers successfully maintained 12 tumor lines from 10 subjects through multiple rounds of serial transplantation. Analyses suggested the likelihood of success wasn’t related to the amount of tumor transplanted or the tumor-to-stromal tissue ratio in the samples.
Interestingly, triple-negative breast cancers tumors (i.e., those that were ER negative (ER−), progesterone receptor negative (PR−), and HER2 negative (HER2−)), usually grew the fastest, which is a phenomenon often observed in the clinic, the team notes. Importantly, evaluation of stained tumors demonstrated that all the grafts retained the major characteristics of the original tumors, even after multiple passages in mice.
Staining and antibody assays indicated that human stroma was largely lost after engraftment and was replaced by mouse stroma including mouse leukocyte, fibrobalsts, and endothelial cells. Importantly ER+ tumor grafts retained estrogen dependence for tumor growth, mirroring a key physiological characteristic of ER+ breast tumors in humans.
The majority of tumor grafts were in addition metastatic and produced patterns of metastasis similar to those of the patients from which the tumor samples were originally taken. The lymph nodes represented the most common site of metastasis in both humans and mice, and while the experimental animals had their inguinal lymph nodes removed during mammary fat pad clearance assosicated with grafting, metastases developed in the thoracic and mesentric nodes, and also in thymus, lungs, bones, and peritoneum. Of notable interest was the fact that bone metastases developed in animals engrafted with tumors from patients either with bone metastases, or whose tumor types are known to metastasize to the bone.
“We surmise that the high metastatic potential of our tumor grafts is related to lack of in vitro manipulation, although this hypothesis remains to be tested,” they note. "Direct implantation of tumors may preserve the ability of cells to interact with supporting cells within the microenvironment; such features may be lost in the in vitro setting. Another possibility is that tumor-initiating cells may be better preserved by direct implantation.”
Turning their attention to the tumor grafts didn’t grow properly due to necrosis after initial engraftment, the researchers postulated whether the addition of human-derived stromal cells might rectify the problem. They therefore re-implanted grafts together with primary mesenchymal stem cells, and found that in both ER- and ER+ tumor grafts, the addition of MSCs increased tumor growth and resulted in greater tumor vascularization. The human MSCs didn’t appear to contribute directly to the formed blood vessels, as these comprised mouse-derived endothelial cells with human MSCs adjacent to them. The addition of MSCs also correlated with strong ER positivity in ER+ tumors, and while ER was clearly retained after serial passage of ER+ tumor grafts without needing to expose them to human MSCs, higher levels of ER staining were demonstrated in tumors had previously been grown with MSCs even after serial transplantation.
“Together, these data suggest that MSCs have multiple, positive effects on human breast tumor grafts, including enhancement of vascularity and maintenance of ER protein expression,” the team writes. “This is noteworthy, as loss of ER protein with tumor progression or serial transplantation is a common problem with models of ER+ breast cancer...Although the mechanisms by which MSCs support tumor growth are beyond the scope of this report, it is plausible that they do so by enriching the microenvironment of the mouse mammary gland with human growth factors, proangiogenic factors, and/or chemokines that favor tumor growth.”
Genetic analyses, including gene expression profiling for cancer subtype-specific genes sets, and genome-wide SNP analyses, confirmed the genetic similarity of the grafted tumors to their parent cancers. Patterns of copy number variations in the original tumors were also maintained in tumor grafts.
The researchers also looked at whether the likelihood of successful engraftment of a tumor sample in mice was due to the type of tumor. To this end they looked at graft data and clinical outcome information from only those 28 individuals with newly diagnosed breast cancer who did not have detectable metastasis at the time of surgery. Follow-up time for these patients averaged 28 months. The results suggested that the ability of a tumor to successfully graft into the mouse correlated significantly with reduced patient survival.
In other words, the most aggressive tumors were the ones most likely to engraft in mice. This finding, the Huntsman team remarks, has potential to be used as a surrogate indicator for the risk of disease progression, even in patients with newly diagnosed breast cancer and without known metastatic disease.
“Orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis, and prognosis,” the authors conclude. The team is making its bank of tumor grafts available as a resource for new models. “Expansion of our tumor graft collection is under way, as is the generation of sublines containing lentivirally delivered markers with which to follow intravital tumor growth and metastasis.”