A new intronic quantitative PCR method led to the discovery of transformation from human ascites to murine malignancy in a mouse model

Abstract

PurposeTo establish a fast and accurate detection method for interspecies contaminations in the patient-derived xenograft (PDX) models and cell lines, and to elucidate possible mechanisms if interspecies oncogenic transformation is detected.MethodsA fast and highly sensitive intronic qPCR method detecting Gapdh intronic genomic copies was developed to quantify if cells were human or murine or a mixture. By this method, we documented that murine stromal cells were abundant in the PDXs; we also authenticated our cell lines to be human or murine.ResultsIn one mouse model, GA0825-PDX transformed murine stromal cells into a malignant tumorigenic murine P0825 cell line. We traced the timeline of this transformation and discovered three subpopulations descended from the same GA0825-PDX model: epithelium-like human H0825, fibroblast-like murine M0825, and main passaged murine P0825 displayed differences in tumorigenic capability in vivo. P0825 was the most aggressive and H0825 was weakly tumorigenic. Immunofluorescence (IF) staining revealed that P0825 cells highly expressed several oncogenic and cancer stem cell markers. Whole exosome sequencing (WES) analysis revealed that TP53 mutation in the human ascites IP116-generated GA0825-PDX may have played a role in the human-to-murine oncogenic transformation.ConclusionThis intronic qPCR is able to quantify human/mouse genomic copies with high sensitivity and within a time frame of a few hours. We are the first to use intronic genomic qPCR for authentication and quantification of biosamples. Human ascites transformed murine stroma into malignancy in a PDX model.

Challenges and Prospects of Patient-Derived Xenografts for Cancer Research

“…In our study of human ascites to murine stromal oncogenic transformation, no humanmurine cell fusions were found, but a sole human somatic mutation, TP53-D281E, was found in the GA0825-PDX model by WES analysis [31]. This mutation may have played a role in oncogenic transformation, as it is adjacent to R282, one of the most common amino acid alterations in TP53 mutations that fail to bind to DNA, specifically, at the TP53 DNA regulatory sequence [44].…”

“…These observations indicated that human prostate tumors were transformed by human tumor cells into mouse oncogenic cells. We (Jin et al, 2023) reported that human ascitic cells (GA0825) from a GAC patient transformed murine stromal cells into a malignant tumorigenic murine P0825 cell line, in a PDX model, in a time-progressive manner [31]. Human-host oncogenic transformation in the PDXs is summarized in Figure 4A,B.…”

“…The Cytogenetics and Cell Authentication Core (CCAC) at M.D. Anderson Cancer Center found a contamination rate of host cells as high as 39% in cell lines sent for authentication [31]. Several studies demonstrated murine stromal and mixed human/murine cells in prostate cancer, indicating that human cancer cells cross-talked to murine stromal cells [32][33][34].…”

“…Now, accumulating evidence suggests that interactions between tumor cells and host cells in the TME are essential for tumor progression and metastases. [31]. (C) Theories have been proposed regarding oncogenic transformations between tumor and normal cells.…”

“…There was one PDX that had a mix of 95.28% human and 4.72% mouse cells, while a second PDX had only 5.36% human cells and 94.64% murine cells. The latter PDX sample presents an obvious dilemma for in vivo drug treatment studies and preclinical personalized therapies if chosen for experiments [31].…”