Modeling human osteosarcoma in mice through 3AB‐OS cancer stem cell xenografts

Abstract

Osteosarcoma is the second leading cause of cancer-related death for children and young adults. In this study, we have subcutaneously injected-with and without matrigel-athymic mice (Fox1nu/nu) with human osteosarcoma 3AB-OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB-OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB-OS CSC xenografts lacked crucial regulators of beta-catenin levels (E-cadherin, APC, and GSK-3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB-OS-derived tumors expressed high levels of pAKT, beta1-integrin and pFAK, nuclear beta-catenin, c-Myc, cyclin D2, along with high levels of hyperphosphorylated-inactive pRb and anti-apoptotic proteins such as Bcl-2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB-OS tumor xenografts obtained with matrigel co-injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1-integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB-OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1-integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments.

Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells

“…Moreover, the presence of OS stem-like cells has been reported in patient tumors [62] as well as in established human OS cell lines [63]. We have previously shown [11,13,14] that 3AB-OS cells highly express a large panel of stemness-related genes/proteins and that efficiently transdifferentiate in vitro into cells of all the three primary germ layers [12], whereas when 3AB-OS cells were engrafted in nude mice, they potently induced malignant tumors, although preserving multilineage commitment [13]. Here, we show that, after in vitro 3AB-OS differentiation, in each derived cell lineage, p53-R248W/P72R was profoundly down-regulated and after p53-R248W/P72R-knockdown the expression of pluripotent markers (Oct3/4, Nanog, Sox2, nucleostemin and CD133) markedly lowered, suggesting that p53-R248W/P72R might be responsible for 3AB-OS cells pluripotency and self-renewal.…”

“…However, prolonged treatment (about 100 days) of MG63 cells with 3AB induced osteocyte death accompanied by progressive enrichment of a new heterogeneous and stable cell population termed 3AB-OS [11], which have properties (self-renewal and pluripotency in vitro, tumorigenicity in vivo) that indicated them as CSCs [12,13] and allowed their patenting. 3AB-OS cells have been characterized at genetic and molecular level: compared to parental MG63 cells, which have a hypotriploid karyotype with chromosome number ranging from 61 to 66, they are hypertriploid with chromosome number ranging from 71 to 82; they also exhibit 49 copy number variations (gains/losses) spanning almost all the chromosomes, 3,512 dysregulated genes and 189 differentially expressed miRNAs.…”

Matrigel: From discovery and ECM mimicry to assays and models for cancer research

“…BME/Matrigel has been used to increase the take and growth of cancer cells, cancer stem cells, and non-cancer cells [2,[163][164][165][166]. The method is simple in that the tumor cells are mixed in the cold with the BME/Matrigel and injected [20].…”

LncRNA MALAT1 Promotes Cancer Metastasis in Osteosarcoma via Activation of the PI3K-Akt Signaling Pathway

“…Osteosarcoma is the most common primary malignant bone tumor in children and adolescents worldwide [1][2][3]. In most cases, the tumors have metastasized to the lungs in patients diagnosed with osteosarcomas [4].…”