Brain Calipers Pdf Free Download ((HOT))
Linh Swanick
So Logan and co-author Christin Palmstrom, an undergraduate student in biology when the research was conducted, acquired skulls from museums and measured them via two methods: They used CT scans to measure the volume of the inside of the braincase and they used calipers to measure the external skull. The scanned the skulls and then used computer software to calculate the endocranial volume, which, according to Logan, is a common proxy for brain size. The CT scan was the more accurate method for calculating endocranial volume, to which they compared length, width and height measurements the made using the calipers.
While genetically engineered mouse models of breast cancer have significantly contributed to the identification of the roles of specific genes in tumor development, they have a low incidence of brain metastasis and do not fully reflect the disease in humans [8,9]. A number of cell-lines have been developed for preclinical breast cancer brain metastasis research [10]. However, the clinical and biological heterogeneity of human tumors cannot be fully reproduced in cell-lines [11,12,13]. Though brain metastases in mice can be generated by directly injecting cells into the blood circulation through the tail vein or into the heart [14], such injections lead to systemic distribution of cells to other organs besides brain. The intracarotid artery injection method was developed to minimize the spread of cells to areas other than the brain [15]. However, this method requires microsurgery skills, and is complicated by a high post-operative mortality rate.
Comparison of three methods for tumor implantation in mouse brain. Brain metastasis of one triple negative breast cancer patient that had been passaged three times as xenograft in mouse brain was used for this experiment. (A) Three methods were used for implantation of mouse brain with tumor. The methods used forceps and tissue blocks (n = 10), 23 G needle and minced tumor tissue (n = 8), or pipette tip and minced tumor tissue (n = 10). 1 µL volume of tumor that had been passaged three times as xenograft was used for each mouse. Pie charts depict survival rates within 1 day of implantation with the three methods. Examples of coronal sections of brain in magnetic resonance imaging are shown for two mice each for the three methods. The extent of tumors in the images are indicated with arrowheads. Rates of engraftment of implanted tumor after 8 weeks of surgery for tumor implantation are plotted for the three implantation methods. Mice that did not survive surgery are excluded. (B) Tukey boxplots of tumor volumes at 6 weeks in mice with engraftment are shown for the Forceps, Needle, and Pipette methods.
Comparison of gene expression of brain metastasis of breast cancer patient with its implant in mouse brain or mammary fat pad. Brain metastasis of one triple negative breast cancer patient was used for this experiment. Tumor was prepared with the mincing method for implantation (1 µL tumor + 2 µL phosphate-buffered saline) in mouse brain (Brain PDX) or mammary fat pad (MFP PDX) with the Pipette or Forceps method, respectively. Implanted tumors were then passaged three times at the same implantation site before they were harvested for RNA sequencing. (A) Mapping of RNA sequencing reads of PDX samples to reference human and mouse transcriptomes. Gene expression of the brain metastasis and implanted tumors was examined by RNA sequencing. (B) Heatmap of gene expression is shown for the 25,012 genes identified as expressed among the human and PDX tumors. Gene expression values are Z scaled, and gene clustering is unsupervised. (C) Unsupervised hierarchical clustering of gene set variation analysis scores of the three samples for the mSigDb Reactome gene set collection. Dendrogram heights are indicated. For clusterings, cosine distance metric and Ward agglomeration method were used. Sequencing reads that originated from mouse cells in PDX tumor stroma were excluded from the gene expression data to generate the plots in panels B and C.
In general, the engraftment rate for PDX generation varies from 4% to 80% [21,38,39]. With our novel method using a pipette with a 10-µL tip, tumors could be prepared and implanted in mouse brain to achieve 100% engraftment and tumor growth without any post-operative mortality. It has been reported that PDX engraftment rates of primary breast cancer vary greatly depending on the subtype [20,40]. In our study, orthotopic PDXs could be equally efficiently generated for both ER+ and ER- breast cancer brain metastases. Moreover, only one cubic mm of tumor was sufficient for engraftment, and tumor growth was observed within almost all mice brains at one month after transplantation. This is important because it demonstrates that our method can be used to generate PDXs relatively quickly without wasting precious human specimens. Although many reports show that PDXs are genetically stable through multiple passages [16,17,18,19], it has also been reported that their genetic phenotype may deviate from the original tumor over time due to selective pressure [41,42]. With our method, passage number can be reduced by transplanting human specimens directly into the mouse brain with a high engraftment rate. The method utilized inexpensive and readily available tools, and was used on more than 40 mice. In contrast, post-operative mortality was high with a tumor implantation method using fine-point forceps, whereas engraftment rate was low with another method using needle for implanting tumors. We believe that in contrast to the other methods, the pipette tip method that we developed allows for more accurate positioning and smoother dispensation of tumor during the implantation procedure.
In some reports of orthotopic PDXs of brain metastases, needles were used for tumor implantation in the brain and tumors were prepared for implantation by dissociating them enzymatically into a single-cell suspension [43,44]. It has been pointed out that enzymatic digestion of tumors reduces the viability of tumor cells (e.g., [45]). This is in agreement with our study that both engraftment rate and tumor growth rate were significantly reduced by enzymatic digestion. While this could be because of a direct effect of enzymes on cancer cells, the extra time that bulk tumor had to be kept in vitro for enzymatic digestion may have also played a role. Many studies have shown the usefulness of embedding tumors in Matrigel for tumor implantation [22,46]. However, in the current study we observed that the growth of brain PDXs was significantly retarded by Matrigel compared to PBS. We could not find a previous citation of such an observation in the existing literature. The effect that we have observed could be because Matrigel inhibits tumor growth factors in the brain environment.
Both tumor engraftment and tumor growth rates of PDX tumors are known to increase over time with serial passaging. This has been shown for xenografts of primary breast cancer [18,47] as well as other cancers [47]. However, there has been no report in this regard on the behavior of orthotopic or ectopic PDXs of breast cancer brain metastases. In our study, we found that these PDXs also displayed increased growth rate with time.
Alzubi et al. demonstrated RNA sequencing data of human breast cancer tumors grown as PDXs at different sites in mouse and separated it into human (cancer) and mouse (microenvironment) transcriptomic datasets. They showed that the cancer transcriptome within PDX was affected by the microenvironment of the tumor implantation site [49]. Rashid et al. utilized a syngeneic immunocompetent mouse model to show transcriptome differences between breast cancer tumors grown in MFP and subcutaneously [50]. These studies show that the tumor microenvironment can affect gene expression in cancer cells of the tumor. Thus, compared to non-brain PDX, transcriptome of the brain PDX is likely to be more similar to the human brain tumor that is implanted.
A systemic review of 113 phase 3 clinical trials on breast cancer from 2011 to 2017 revealed an overall failure rate of 65% [51]. One of the reasons for this high failure rate is inability to exclude from clinical trials investigational drugs that are not effective in preclinical animal models. Epothilone B is one such drug, whose anti-cancer activity was expected from its taxane-like effect on microtubule stabilization [52]. Though epothilone B has a high anti-cancer activity against primary breast cancer tumors and can cross the brain-blood barrier [36], it failed a phase 2 clinical trial for effectiveness against breast cancer brain metastasis [37]. In our study, while brain metastasis PDXs grown ectopically in mouse MFPs responded to the drug, with tumor growth retarded by about a third compared to untreated tumors, orthotopic PDXs did not respond to treatment. This result suggests that if orthotopic PDXs had been used in preclinical studies of epothilone B, then its inefficiency may have been detected before clinical trials, which could have saved patients from useless therapy and huge expenses as well as emotional turmoil.
The following are available online at -6694/12/2/444/s1: Figure S1: Survival of mice whose brains were implanted with brain metastasis of two breast cancer patients, Figure S2: Histology of breast cancer brain metastases grown as xenografts in mouse brain or mammary fat pad (MFP), Figure S3: Survival of mice whose brains were implanted with brain metastasis of two breast cancer patients, Table S1: Human breast cancer brain metastasis samples used in this study, Table S2: Gene expression data generated in this study.
The first author contacted the journal editors to report an error in the charts shown in Figs 2C and 3C, and provided revised tumor volume charts reporting smaller tumor volumes compared to the published figures. The first author stated that vernier calipers were used to measure tumor volume, and that the errors in the published tumor volume data were found during routine raw data inspection. The underlying individual-level quantitative data for all figures were provided, along with the in vivo imaging system (IVIS) output files underlying the photographs in Figs 2C and 3C and all experimental replicates, and a copy of the ethical approval document with reference number NCC2014GZ-01.
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