Human tumor cell lines are extremely important tools for cancer research, but a significant percentage is cross-contaminated with other cells. Short tandem repeat (STR) profiling is the prevailing standard for authenticating cell lines that originate from human tissues. Based on the analysis of 482 different human tumor cell lines used in China by STR, up to 96 cell lines were misidentified. More importantly, the study has found that STR profiling alone is insufficient to exclude inter-species cross-contamination of human cell lines. Among the 386 cell lines which had a correct STR profile, 3 of them were inter-species cross-contaminated. Careful microscopic examination may be helpful in some cases to detect changes in morphology but additional testing is needed. Additionally, species verification by PCR could easily identify the contaminants, even with a low percentage of contaminating cells. Combining STR profiling with species identification by PCR, more than 20.5% (99/482) of tumor cell lines were revealed as having been incorrectly identified, including intra-species (14.5%), inter-species (4.4%) cross-contamination and contaminating cell lines (1.7%). Therefore, quality control of cell lines is a systemic issue. Each cell line should undergo a full QA (Quality Assurance) assessment before it is used for research.

CD133 has been identified as a cancer stem cell marker in colon and several other cancers, but its function is still unknown. We examined the CD133 expression in 44 human cancer cell lines, and found five of the 8 positive lines were from colon cancer. The CD133 positive subpopulation of colon cancer cells showed more vigorous growth and lower differentiation. Induction of differentiation reduced the CD133-positive population. Knockdown of CD133 expression in colon cancer cells could not induce cellular differentiation. Care must be taken if CD133 is used as the only marker of cancer stem cells in colon cancer, especially in established cell lines. CD133 negatively correlates with cell differentiation, but it is not a regulator of differentiation.

CD133 is widely expressed in colorectal cancer (CRC) tissues and cell lines. This protein has been used as a marker of CRC cancer stem cells, although the function and mechanism of CD133 in CRC invasion and metastasis remain unclear. In our study, we examined the role of CD133 in CRC invasion in vitro and investigated the mechanism involved in CD133-related invasion. CD133(high) and CD133(low) HCT116 cells were isolated, and the proliferation and invasive ability of these two subpopulations were tested. CD133(high) HCT116 cells exhibited greater proliferation and invasion compared with CD133(low) HCT116 cells. CD133 knockdown (using CD133 small-interfering [si]RNA) inhibited the invasive activity of CD133si-HCT116 cells. For the first time, we found that the expression of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was down-regulated in CD133si-HCT116 cells. In addition, for the TIMP-2si-HCT116 cells (transfected with TIMP-2 siRNA), in vitro invasion was significantly decreased, whereas the expression of CD133 remained unchanged. Finally, the metalloproteinase 2 and MEK/ERK signaling pathways were examined, and no significant change was observed after the knockdown of CD133 or TIMP-2 in HCT116 cells. In conclusion, we demonstrated that CD133 plays an important role in HCT116 cell invasion, and for the first time, we found that CD133 knockdown significantly down-regulated TIMP-2 expression, which suggests that CD133 affects the invasive ability of HCT116 cells by regulating TIMP-2.

Human tumor cell lines, especially those with complete data and follow-up, are important tools in tumor biological studies. Clear cell renal cell cancer (ccRCC) is not sensitive to radiotherapy or chemotherapy, and treatment of patients with distant metastasis relies on targeted therapy. Here, we report the establishment of seven new ccRCC stable cell lines that were continuously cultured for more than 20 generations among 81 cases of renal cell cancer. Moreover, gene expression and methylation in the established cell lines, in those that had a finite in vitro life span of less than 10 generations, and in cells that originated from the same culture at different generations were profiled using microarrays. Genes including SLC34A2, SEPP1, SULT1C4 and others were differentially expressed in established cell lines and finite cell lines, and changes in their expression might be caused by methylation or demethylation. The expression level of SLC34A2 was related not only to the life span in vitro culture but also to tumor size. Additionally, six of the seven new ccRCC cell lines had VHL deletions or termination mutations. So in addition to the establishment of seven new ccRCC cell lines with complete clinical data, we conclude that genes such as SLC34A2 and VHL play key roles in the continuous in vitro growth and development of ccRCC.

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The short‐tandem‐repeats (STR) profiles of MGc80‐3 and HeLa partially overlap, raising suspicion of contamination in the MGc80‐3 cell line. However, there has not been any relevant study demonstrating whether MGc80‐3 was fully replaced by HeLa cells, just mixed with HeLa cells (co‐existing), or was a somatic hybrid with HeLa cells. In addition to STR profiling, various approaches, including single nucleotide polymorphisms genotyping, polymerase chain reaction, screening for human papillomaviruses type 18 (HPV‐18) fragment, chromosome karyotyping, pathological examination of xenografts, tissue‐specific‐90‐gene expression signature and high‐throughput RNA sequencing were used to determine the nature of MGc80‐3. Our study found that the abnormal STR profile, partially overlapping with that of HeLa cells (64.62% to 71.64%), could not verify MGc80‐3 as a HeLa cell line. However, the STR 13.3 repeat allele in the D13S317 locus that seemed to be unique to HeLa cells was detected in MGc80‐3. Almost all the MGc80‐3 cells exhibited HPV‐18 fragments in the genome as well as certain HeLa marker chromosomes, such as M7 and M12. The molecular assay of the 90‐gene expression signature still considered MGc80‐3 as a stomach cancer using an algorithmic analysis. The expression pattern of multiple genes in MGc80‐3 was quite different from that in HeLa cells, which showed that certain characteristics belonged to gastric cancer cell lines. High throughput RNA sequencing showed the distinct patterns of gene expression in MGc80‐3. In conclusion, MGc80‐3 cell line is a somatic hybrid with HeLa cells rather than a pure gastric cancer cell line.

Backgrounds: Epidermal growth factor (EGF) is a 53 amino acid polypeptide and its receptor EGFR is an established therapeutic target for anti-tumor therapy. Two major categories of EGFR-targeted drugs include monoclonal antibodies (mAbs) and small molecular tyrosine kinase inhibitors (TKIs). However, drug resistance occurs in a significant proportion of patients due to EGFR mutations. Since EGFR can maintain activation while abrogating the activity of mAbs or TKIs, or bypass signaling functions while successfully circumventing the EGF-EGFR switch, developing new mechanism-based inhibitors is necessary. Methods: In this study, based on the principle of tumor immunotherapy, a recombinant protein pLLO-hEGF was constructed. The N-terminal portion contains three immunodominant epitopes from listeriolysin O (LLO) and the C-terminal includes EGF. To use EGF as a target vector to recognize EGFR-expressing cancer cells, immunodominant epitopes could enhance immunogenicity of tumor cells for immune cell activation and attack. Results: Recombinant protein pLLO-hEGF was successfully expressed and showed strong affinity to cancer cells. Also, pLLO-hEGF could significantly stimulate human lymphocyte proliferation and the lymphocytes demonstrated enhanced killing potency in EGFR-expressing cancer cells in vitro and in vivo. Conclusion: This study can provide novel strategies and directions in tumor biotherapy.

Abstract. SRS19-6MuLV is a member of the MuLV family originally isolated from the Tianjin-Shanghai-Zunyi complex of murine leukemia. A notable characteristic of this virus is that it induces tumors of multiple hematopoietic lineages, including myeloid, erythroid, T-lymphoid and B-lymphoid. In a previous study, a sequence with high homology to SRS19-6MuLV in a murine dendritic cell sarcoma (DCS) was identified through cDNA expression screening with mAb 983D4. To investigate the relationship between SRS19-6MuLV and DCS, the existence of a specific SRS19-6MuLV DNA fragment in DCS cells, 15 murine tumor cells, 2 murine tumor tissues, 12 normal murine cells/tissues, 11 human tumor cell lines and SRSV/3T3 (NIH/3T3 cells infected with SRS cell supernatant) was detected by PCR. The specific fragment of SRS19-6MuLV was detected in DCS, mouse fore-gastric cancer cells, LⅡ tumor tissue from which DCS is derived and SRSV/3T3. In addition, the integration sites of SRS19-6MuLV in the positive cells were examined by inverse PCR. Thus, 7 integration sites for SRS19-6MuLV were detected in DCS and 3 in SRSV/3T3. Analysis of sequences by BLAST revealed that some of the integration sites were associated with common fragile sites and some Ras-regulating miRNAs. Our results indicate that SRS19-6MuLV not only induced four types of leukemia, but also induced DCS. This virus does not infect human cells. Multiple integration of SRS19-6MuLV into chromosomes around fragile sites accounts for its carcinogenic effects.