Title: Analysis of the nuclear morphological characteristics of MCF7 human breast cancer cells in culture in comparison to CACO2
Introduction: Breast cancer is a severe global health predicament being the 2nd most frequent of and by far the most regular cancer affecting women (Sonnenschein, 2010). The resistance development to chemotherapeutic elements or agents is a restrictive factor in the neoplastic diseases management and controlling results to treatment failure. Even though there is indication that the reflective metabolic transformation brings structural and morphological cellular alteration, these changes are not particular or vital adequately to be dependably recognized under the microscope. Deoxyribonuclei acid (DNA) structural changes and development of resistance in some cell lines recommend that scrutiny of nuclear morphology could be useful in separation of drug receptive and resistant cells (Sonnenschein, 2010). The nucleus is sculpted towards different morphologies throughout tcellular development and differentiation process. Transformation in nuclear shape, usually result in alteration to chromatin arrangement and genome purpose. This is believed to be reflective of its responsibility as a cellular mechano transducer (Sonnenschein, 2010). I only put one reference here. I want you to add one or two more different references.
Eukaryotic cells contain a nucleus which is responsible for transcriptional regulation (Tokunaga K et al 2014). Current research has shown that there are molecular changes that take place in cancer cells and these changes are often associated with result in deranged cellular functions of a cancer cell (Dahl et al. 2008). Therefore, the morphology of nucleus of cancer cells is of prime interest to cytopathologists. According to Zink et al. (2004), the nuclear changes in biopsy samples have remained the most accurate and standard method of diagnosing cancer. The nucleus of the cancer cell has been shown to exhibit characteristic microscopic changes such as alteration in nuclear size shape margin, chromatin pattern, nucleoli and perinuclear space. Cancer cells typically have a nucleus larger than that of a normal cell and looks darker when seen under a microscope (Schneider et al 2007). Nuclear shape and margin irregularity is common in cancer. Nuclear margin irregularity is seen as nuclear grooving, nuclear molding, and nuclear convolutions. During cell differentiation, nuclear structures are reconfigured dynamically. Previous studies have identified numerous distinct nuclear bodies. The perinucleolar compartment (PNC) accumulates polypyrimidine tract binding protein and several polymerase III RNAs, which has been shown in virtually all types of solid tumours (Tokunaga K et al 2014). The nuclear membrane (NM) proteins that contribute to the preservation of nuclear shape and its organization have been demonstrated to be tissue specific, and are altered in many cancers.
The objective of this research is to ascertain and analyze the unique characteristics of nuclear structural features and composition. Some of these structural elements include the size, form, and margin of the nucleus. Other features include the irregular nuclear shape, chromatin outline, anaphasic bridges, nucleoli and micronuclei. As the changes in the nuclear organisation and morphology might be tumor specific, this study aims at comparing the nuclear parameter between two different types of cancer cells- MCF7 and CACO2. (Make this paragraph more specific and bring it to an appropriate place where it looks suitable.)
Breast cancer understanding:
This is the paragraph taken from the ‘fragile site report’. The level of CFS occurrence has yet not been critically evaluated within malignant neuroblastoma cells. Therefore, this study is aimed to determine the fragility retained by the established cell line of SH-SY5Y human neuroblastoma. The SH-SY5Y cell line represents the third consecutive sub-clone of the SK-N-SH line, initially taken from metastatic neuroblastoma patient via a bone marrow biopsy. As differentiated neurons do not divide, the malignant SH-SY5Y neuroblastoma cell line can be broadly utilised in-vitro for its dividing characteristics. Potentially, this cellular property has permitted the SH-SY5Y cells to be used for research in neurodevelopmental and neurodegenerative disorders.
Now you have to write a similar sort of paragraph for MCF7 mentioning from where it is extracted to establish the cell line and other related bits as my friend mentioned in her report (here in blue bold above paragraph)
Colorectal cancer understanding:
And a separate paragraph mentioning the same sort of thing for CACO2.
Methodology:
Cell culture:
Human cancer cell lines MCF7 and CACO2 cell lines were obtained from European collection of cell cultures. The culture initiation consisted of a seeding density of approximately 1 × 105 cells per milliliter which was utilized for a single T25 flask having ten milliliter of cell growth solution [1:1 Dulbecco’s Modified Eagle Medium (DMEM) + 10% (v/v) FBS]. The doubling period for cells was roughly twenty eight hours (this is grounded on the computations of haemocytometer using trypan blue, prior to passage II). MCF7 cells were sub-cultured to achieve 85-90% confluency in T25 flask. However, the procedure for proliferating CACO-2 contrasts from standard sub-culturing procedure mainly because CACO-2 cells are passaged when they achieve just 50% of confluence, instead of 85%, retaining a high proliferation potential.
The thawing procedure involved decanting out DMEM from the previous pvalu and washed with PBS (phosphate-buffered saline) free from Ca2+ and Mg2+ at PH 7.4. The cells were harvested from the adherent monolayer of cells using EDTA (versene) solution of 0.526 mM (1:5000) at cell growth conditions of 37 degrees Celsius in moistened, concentrated Carbon (IV) Oxide (5%) atmosphere. Cells were then re-suspended in the fresh DMEM to stop the enzymatic action of trypsin and their viability assessed using 0.4 % trypan blue solution to determine cells seeding density. The cells were transferred into conical tubes and centrifuged at 800rpv (revolution per minutes) for five minutes. The visible white cell pellet was separated after aspirating out the supernatant and pellet was dislodged and resuspended in the fresh DMEM at a ratio determined by trypan blue assessment and was left to incubate for approximately one week. All reagents used in this study are manufactured by Gibco® and can be accessed through life technologies.
Fixation??: The cells were treated with hypotonic medium of 0.55% Potassium Chloride (KCl) solution using drop by drop technique and nurtured for five minutes at room temperature so as to permit the cells swell in this hypotonic medium. Cells were centrifuged for 8 minutes at 1200rpv. 3:1[methanol (CH3OH) : acetic acid (CH3COOH)] solution was used to fix the cells. Cells were left at room temperature for five minutes and centrifuged for another eight minutes at 1200rpv. The slides drenched in ethanol, dried and used to fix the cells by pipettes. Four slides were prepared for each human cancer cells MCF7 and CACO2.
Giemsa staining The Giesma working stain used had been made and placed in Coplin jar. Slides were placed in the Coplin jar during the staining stage. Slides were constantly monitored to avoid over staining. The stained slides were washed in the two changes of distilled water before being left to dry in the racks in preparation of getting affixed with DPX ready for microscopic examination.
Microscopic analysis: Axioskop 2’ microscope fitted with “Axio high-resolution 3 digital cameras were leveraged to take images of the CACO2 and MCF7 nuclei. The microscope objective lens had been set up as ×100, although the sum magnification totaled up to ×1000 as the eyepiece lens enlarged the image by ×10. Zeiss immersion oil was used to eliminate refractive properties of the glass used in the slide while AxioVision software had been fitted in the camera and computer so as to allow for graphic interpretation of several metamorphic analyses. AxioVision software was set to a resolution of 277 × 2080 pixels, thereby allowing for capture of images that would facilitate accurate analysis.
Morphometric analysis: Nuclei were stained with Giemsa and pictures were taken on an Axioscope 2 microscope for morphometric analysis. ~200 nuclei were analysed in random area of fields for each of MCF7 and CACO2 using ZEISS axioskop 2 microscope. The morphometric analysis was performed using the graphic tablet (Wacum intuos 15.2 x 9.5cm) and ImageJ software (Microsoft Java) downloaded from imagej.nih.gov/ij/. The nuclei were selected and outlined. After outlining the nucleus boarder, the information was analysed. The information analysed were nuclear area (NA), nuclear perimeter (NP), and nuclear contour ratio (Contour ratio = 4 pi nuclear area/ nuclear perimeter2) and their respective units and SDs were assessed using the image analysis. The area was measured in µm2 whereas perimeter in….. This data was used for the statistical analysis of measurements of main significance for morphometric analysis.
STATISTICAL ANALYSIS: SPSS software package (IBM® SPSS® Statistics V22.0) was used for the statistics analysis. The student sample t-test was used to compare the mean of the area, perimeter, nuclear contour ratio and number of nucleoli/nucleus of the two abnormal epithelial cells from the colon (CACO-2) and breast (MCF7) by SPSS Inc. Pearson test (Chi square test) was used to compare the other nominal variables (irregular shape, nuclear bleb, anaphasic bridges and micronucleus). In this report, all the student sample t-test and Pearson Chi sqaure tests were performed by SPSS software. All the values are presented as a Mean (M), standard error of mean (SEM), Standard deviation (SD). All the values are rounded up to the nearest whole number and to 2 s.f. where applicable. For all the statistical tests, the results were considered significant at p < 0.05. In this report, all the results were statistically significant at p < 0.05 and p < 0.001, and at the significant level of 0.05 (5%) meaning that all the results were statistically real and not due to chance.
RESULTS:
Random fields of slides were selected to determine the nuclear characteristics of MCF7 and CACO2 with Axioskop 2 digital microscope. A total of approximately 200 nuclei were observed for each of MCF7 and CACO2 to determine their nuclear characteristics. The nuclear features of MCF7 and CACO2 that were analysed statistically were nuclear shape irregularity, presence of nuclear bleb, existance of anaphasic briges, nucleoli, micronucleus…….
NUCLEAR SHAPE
The MCF7 have significantly higher number of abnormal shaped nuclei (figure 4) and nuclear protrusion (figure 5) when compared with CACO2 nuclei. The nuclear shape irregularity was assessed quantitatively after measuring the individual nuclear cross-sectional area and perimeter of each of MCF7 and CACO2 nuclei. The nuclear cross-sectional area and perimeter was used to compute the nuclear contour ratio (4π x area/perimeter2), which provides a quantitative measure of nuclear circularity. Contour ratio for the circular shape has a value of 1, whereas more convoluted outlines lead to smaller values. This report demonstrates that MCF7 cells has a significantly lower mean contour ratio compared to CACO2 cells (Figure 3a). and relative frequency distribution (figure 3b) represents that MCF7 cells has an overall distribution shifted slightly more towards the contour ratio values (X-axis), which indicates the presence of greater number of abnormally shaped nuclei compared to CACO2 (Figure 3b). According to Lammerding et al (2005) & (2006), the normal shaped nuclei has a contour ratio of ~ 0.90. Figure 3b also demonstrates that the number of MCF7 cells reaching the normal value (which is approximately 0.90) is significantly lower than CACO2. However, CACO2 shows a wider distribution of nuclei, with more cells/nuclei present at the higher value while a small proportion of cells also present at the lower values of nuclear contour ratio. The cross-sectional area and perimeter for MCF7 was significantly lower compared to CACO2 (Figure 1 and 2), indicating that lower nuclear cross-sectional area and perimeter has significantly higher contribution to the nuclear shape irregularity.
Figure 1- Morphometric analysis of area (a) and perimeter (b) of the nuclei of MCF7 and CACO2. The area and perimeter are demonstrated as bar graphs. The area was measured as µm² whereas perimeter in… a) Data set was analysed by independent sample t test using SPSS which demonstrated that there is significant statistical difference between the nuclear area of the two cancer cells MCF7 and CACO2 (p < 0.05). The mean nuclear area of CACO2 (M = 198395, SEM = 9111, SD = 80472) is significantly higher than MCF7 (M= 82668, SEM = 2370, SD = 36573). Error bars are SD. B) Findings of the Independent sample t test demonstrated that there is significant difference between the nuclear perimeter of MCF7 nuclei (M= 1133, SEM = 21, SD = 328) and CACO2 nuclei (M = 1649, SEM = 39, SD = 346) conditions; t (407) = -11.8, p < 0.001. The mean nuclear perimeter of CACO2 is significantly higher relative to MCF7.
Figure 2- Morphometric analysis of the nuclear contour ratio for MCF7 and CACO2. a) Represents the comparison of mean nuclear contour ratio of MCF7 and Caco2. The independent sample t-test was performed using SPSS software to find the statistical relationship between the Nuclear contour ratio and Cancer cells-MCF7 and CACO2. The results showed that there was a significant difference between nuclear contour ratio of MCF7 (M = 0.81, SEM = 0.0081, SD = 0.12) and CACO2 (M = 0.90, SEM = 0.010, SD = 0.088), conditions t (407) = -6.253, p < 0.001 (Independent sample t-test). Nuclear contour ratio is significantly lower for MCF7 nuclei compared to CACO2 nuclei.
Figure 3: Demonstration of relative frequency distribution of the contour ratio for MCF7 and Caco2. The median values for MCF7 and Caco2 were 0.0803 and 0.889 respectively.
Irregular shape
Figure 4: Arrows demonstrate the nuclei which indicates the mild deviation from the normal circular shape (contour ratio < 0.90) as measured by the ImageJ software. Yellow (contour ratio = 0.78), Red (contour ratio = 0.79), Blue (contour ratio = 0.82).
Figure 5: Graphic representation of irregular shape. The figure represents the number of abnormally shaped nuclei of MCF7 and CACO2 determined microscopically in the range of 200 nuclei analysed. Visual analysis and nuclear contour ratio (index value < 0.90) was conducted to evaluate the irregular shape. Statistical analysis with Chi square test demonstrated that there is a significant difference between the irregular shape of MCF7 and CACO2 X2 (df = 1, N = 400) = 32.7, p < 0.001. The number of irregular shape for MCF7 is statistically higher than CACO2 nuclei.
Nuclear bleb:
Figure 6- Represents the nuclear bleb captured by ZEISS Axioskop 2 microscope (Giemsa stain at 100x objective). A) Demonstrating the CACO2 nucleus with chromatin protrusion of nucleus (nuclear bleb) and B) showing the MCF7 nucleus with nuclear bleb.
Figure 7: Demonstrate the Nuclear bleb in MCF7 and CACO2. Pearson Chi square test was performed on SPSS software which represented that there is a significant difference (p < 0.05) between the nuclear bleb of MCF7 and CACO2, X2 (df = 1, N = 407) = 7.06, p = 0.008. The number of nuclear protrusion for MCF7 is significantly higher (22) compared to CACO2 (8 nuclear bleb).
Anaphasic bridges
Figure 8:- a) and b) Demonstrating the formation of anaphasic bridges (Giemsa x 100 objective). 3a) was captured with the axioskop 2 digital microscope while 3b) was captured on the simple light microscope (x100 objective).
Figure 9: Represents the percentage of Anaphasic bridges in MCF7 and CACO2. A total of 4 anaphase bridges observed in MCF7 cells whereas none is observed in CACO2 under the conditions tested. Pearson Chi square test (computed with 2×2 contingency table) was performed on SPSS software to analyse the significant relationship between the anaphasic bridges of cancer cells (MCF7 and CACO2). Statistical analysis with Chi square test demonstrated that there is a slightly significant difference between the anaphasic bridges of MCF7 and CACO2 X2 (df = 1, N = 170) = 4.46, p = 0.044.
Micronuclei
Figure 10: represents the different types of MCF7 cells with micronucleus captured with axioskop 2 digital microscope (Giemsa x100 objective). a) represents a micronucleus associated with a binucleated cell and b) shows various sized micronuclei (arrows) which is not associated to any of the surrounding nuclei.
Figure 11: Represents the percentage of Micronucleus in MCF7 and CACO2. A total of 4 anaphase bridges observed in MCF7 cells whereas none is observed in CACO2 under the conditions tested. independent sample Mann-Whitney U test was performed the distribution of micronucleus is same across the catagories of cells.
Nucleoli:
A little bit about nucleoli:
Figure 12- Represents the mean number of nucleoli per cell/per nucleus in MCF7 and Caco2. The data set was analysed using independent sample t test on SPSS and the results demonstrated that there is significant difference between the number of nucleoli of MCF7 (M = 2.90, SEM = 0.067, SD = 1.03) and CACO2 (M = 1.95, SEM = 0.083, SD = 1.08), conditions t (405) = 9.02, p < 0.001. The number of nucleoli for MCF7 (~3) was significantly higher relative to CACO2 (~2 nucleoli/nucleus).
Table 1: An overview of the statistical test
Category Subcategory Statistical test Statistics value i.e X2 value or t-value p-value
Morphometric feature by ImageJ Area Independent sample t-test < 0.001
Perimeter Independent sample t-test -11.8 < 0.001
Contour ratio (circularity) Independent sample t-test -6.253 < 0.001
Irregular shape Pearson chi-square 32.7 < 0.001
Nuclear bleb Pearson chi-square 7.06 0.008
Anaphasic bridges Pearson chi-square 4.46 0.044
Nucleoli Independent sample t-test 9.02 < 0.001
Micronuclei Pearson chi-square
The individual results of each nuclear feature were statistically analysed by conducting statistics tests (t-statistics and Chi-sqaure test) at significance level (alpha) of 0.05, which allowed a proportionality comparison of the nuclear features of MCF7 and CACO2. Statistical tests were performed to compare eight nuclear characteristics of MCF7 and CACO2. All the results are statistically significant at a p-value of less than 0.05 and 0.001 and at a significant level (α) of 0.05 which infers that the results were statistically real and not due to chance. The statistical tests values (t-test and Chi square test values from table 1) also proved that there is a significant difference in nuclear characteristics of MCF7 and CACO2. Thus, the hypothesis of this study stating, ………………….. is accepted to be true. Null hypothesis (Ho)
Discussion:
Limitation:
Future work:
Discussion:
Abnormal nuclear morphology is one of the hallmark of neoplasia. Indeed, nuclear morphology is one of the features that can be used for the grading and staging of cancer from histopathological point of view in particular Breast cancer and colorectal cancer. In this study, the difference in the nuclear morphology of two types of cancer were compared. The results discerned that both are abnormal but there were substantial differences. There was a significantly higher nuclear abnormality observed in MCF7 cells relative to CACO2 cells. Evaluating Table-1, P-value for all the observed nuclear features was less than 0.05 and 0.001 that the probability of the results due to chance is insignificant. The statistical tests values (t-test and Chi square test values from table 1) also proved that there is a significant difference in nuclear characteristics of MCF7 and CACO2. In MCF7 and CACO2 nuclei analyzed by Giemsa, the vast majority of irregular shape, nuclear blebs, anaphasic bridges, nucleoli and micronuclei were from MCF7 whereas a small to moderate proportion (TABLE-1) of abnormal nuclear structures were from CACO2. Thus, the hypothesis of this study stating, the nucleus of cancer cell exhibit specific dissimilarities in nuclear structural traits and composition is accepted to be true.
An abnormal nuclear shape is also associated with cancer (Zink et al., 2004). In fact, altered nuclear shape is one of the key diagnostic tools used in identifying cancerous cells (Webster et al, 2009). (Kindly paraphrase these two sentences taken from https://www.researchgate.net/publication/24406286_Sizing_up_the_nucleus_nuclear_shape_size_and_nuclear-envelope_assembly_J_Cell_Sci ). In this study, morphometric analysis of nuclear contour ratio (figure 3a & b) was used to assess the irregular nuclear shape. Statistical analysis of irregular nuclear shape (figure 4) and nuclear bleb (THIS COMES UNDER IRREGULAR SHAPE I GUESS, DOUBLE CHECK). confirmed that both MCF7 and CACO2 nuclei exhibited a significant number of irregular nuclear shapes (65% for MCF7 and 39% for CACO2). However, MCF7 were found out to contain more distorted nuclear shapes than CACO2 cells (figure 4). In comparison, you can say about the nuclear shape of normal cell which is round (find reference) and then you can score them and grade etc. Hope you understand.
So far, I was looking on these two websites
http://pathology.jhu.edu/breast/grade.php
https://www.iarc.fr/en/publications/pdfs-online/pat-gen/bb2/bb2-chap6.pdf
But Let me remind you that this is Nuclear pleomorphism, (NOT CELLULAR)..shape will be linked to grading then for other bits nucleoli, micronucleus ,anaphasic bridges you can link it to say that since the MCF 7 proved to be at lower grader then you can also bring your discussion how these results are different from each other you can also discuss what went wrong. Same goes for other stuffs, you can also talk about genetics like what went wrong or other relevant stuff in terms of cancer but don’t go off the track. Also you can make it more scientific and one very important thing that you can discuss is the results in this report whether they are consistant or inconsistent with the literature if possible.
You can also check the criteria for scoring and grading for nuclear pleomorphism. Like nucleoli, shape. I am not sure what can you say about the micronuclei and anaphasic bridges. But link the shape and nucleoli with the grading, staging or scoring whatever it is.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1886650/?page=1
http://clincancerres.aacrjournals.org/content/5/11/3542.full
http://www.ncbi.nlm.nih.gov/pubmed/20434415
http://jcb.rupress.org/content/170/5/781.full
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1850274/#b1
