More than 90% of of cancer related mortality is caused by metastasis. Todevelop new therapeutic strategies it is vital to understand the initiation andprogression of metastasis. To identify and isolate metastasis initiatingtumour-cells scientists developed a fluorescence-activated cell sorting(FACS)-based array.
There are two types of metastasis-cells: metastasis-cellsfrom low-burden cells and metastasis-cells from high-burden cells. After beingtransplanted, low-burden metastasis-cells showed that they have a considerableamount of tumour initiating ability and could differentiate to produceluminal-like cancer cells. When low-burden metastatic cells progress tohigh-burden metastasis cells they get an increased proliferation and MYCexpression. This can be weakened with the use of inhibitors. This all supportsa hierarchical model in which metastasis is initiated by stem-like cells, andcan progress from low-burden to high-burden metastasis cells. The human breast contains two kinds of epithelial lineages: thebasal/myoepithelial which contains stem-cells, and the luminal lineage whichcontains progenitor cells and mature cells.
The scientists used breast tissuefrom the mammoplasty of three individuals. With the use of numerous statisticalanalysis they concluded that basal/myoepithelial and luminal lineage isdifferent for everyone.They focused in this experiment on a particular subtype,because this subtype is the most aggressive and there is no suitable treatmentfor it. The patient-derived xenograft maintained the same properties in themice as in the patients and because of this it was suitable for the studies ofhuman metastasis. To isolate the metastasis cells from the patient derived xenograft micethey developed a new FACS based array. With this they were able to detectmetastatic cells in 70% of the patient-derived xenograft peripheral tissue fromthe mice.
The mice were analyzed when their tumours reached 20-25 mm indiameter. The growth kinetics was consistent within each model. Although the tumour of every animal had about the same diameter they allhad great variation in metastatic burden. The scientists also found out thatPCA plots for mice with low burden metastasis cells were further away from thetumour they derived from than high burden metastasis cells.
Other experimentsalso showed that low burden metastasis cells liked to form clusters with eachother while high-burden metastasis cells liked to form clusters with primarytumour cells. The scientists found out that low burden metastasis conservedtheir basal/myoepithelial signatures. They had expressed higher levels of 22basal/myoepithelial genes and expressed lower levels of 7 luminal genes.
By focusing on clustering only the metastasis cells the scientistsdiscovered incredible heterogeneity in differentiation, which correlated withmetastatic burden. Akin to the mammary gland metastatic cells organized intotwo different clusters, where the low-burden cells were the mostbasal/stem-like and the high-burden cells were the most luminal-like. The scientistsconcluded the same conclusion with lung metastatic cells. Which means that itis a conserved phenomenon in each model.
There were some differences betweengene expression of lung metastatic cells of different models, but they were notenough to cluster metastatic cells separately by patient derived xenograftmodels. In order to investigate heterogeneity at protein level scientists performedimmunostaining for a basal and luminal gene. Tumour cells found inmicrometastatic from low-burden tissues had a high percentage for the basalgene and luminal gene and tissues from high-burden tissues had a highpercentage for the luminal gene and were heterogeneous for the basal gene. Thissuggest that differentiation status correlates with metastatic burden inprotein-level. By means of single cell analyses scientists discovered that in thelow-burden metastatic cells had high levels of pluripotency genes. These genessuggest that they are exploited embryonic programs for self-renewal andmaintenance. Low-burden metastatic cells also expressed higher levels oftypical EMT-markers, except for an EMT-marker which was typically found innormal basal/stem cells. All these findings are consistent with previousreports which show that EMT promotes stemness in mammary gland, and suggestthat low-burden metastatic cells utilize an EMT-program to make disseminationeasier.
Further studies also revealed that genes involved in the DNA damageresponse, chromatin modification, differentiation, apoptosis and the cell cyclewere differentially expressed in low-burden metastatic cells. Because of the heterogeneity in metastatic cells scientist wondered ifstem-like cells directly give rise to luminal-like cells, or if the luminalcells are originated from founder-cells. After an experiment the scientistconcluded that luminal-like cells can derive from cells that disseminate at theearly stages of primary tumour growth. To test the growth and differentiation capacity of stem like metastaticcells, scientists transplanted low-burden metastatic cells into mammary glands.
Interestingly 2 of 4 transplanted cells produced large tumour, while primarytumour cells never produced tumours, even at 100 fold high numbers. This isconsistent with the previous reports which showed that PDX tumours are moreefficiently increased as fragments than dissociated cells. After single cellanalyses scientists concluded that low burden-metastatic cells have high tumourinitiating capabilities, and that they can give rise to luminal-like tumourcells. This supports the hypothesis that stem-like metastatic cells give riseto luminal metastatic cells. Another interesting question the scientists had was is stem-like cells werepresent in tumour cells, or if they evolve after interaction with theirmicroenvironment. After a test the scientists concluded that primary tumourscontain a rare subpopulation of stem-like cells, and that the percentagecorrelates with metastatic potential. Afterwards scientists wanted to know ifenrichment of this stem-like signature in primary tumours may be predictive ofdistant metastasis in human patient data sets. After an analyses the scientistsfound that 16 of 55 genes associated with stem-like metastatic cells weresignificantly prognostic.
Previous work has shown that metastatic cells in different organs displaydistinct gene expression signatures. Supervised clustering by target organ hasshown that metastatic cells in brain, bone marrow and peripheral blood haddifferences in gene expression patterns. Brain metastatic cells were the mostdistinct. CTC’s are very important for use as a ‘liquid biopsy’ for diagnosis andprognosis. Most CTCs and bone marrow DTCs clustered with ‘intermediate’metastatic cells, which may be because the cells were harvested from animalswith intermediate burden.
However, 16.7% and 10.7%, showed a morebasal/stem-like signature, suggesting that these stem-like cells may representthe true metastatic seeder cells. We also observed a shift towards a more proliferative signature associatedwith increased metastatic burden. Low-burden metastatic cells expressed higherlevels of rest and dormancy-associated genes. Higher-burden metastatic cellsappeared to enter the cell cycle, expressing lower levels of quiescence and dormancy-associatedgenes and higher levels of cell-cycle-promoting genes.We also detected primarytumour cells (22.2%) with this less-proliferative signature.
These findings prompted us to test whether blocking this switch fromdormancy into the cell cycle could inhibit metastatic progression. Since weobserved high levels of both MYC and CDK2 in more advanced stage metastaticcells , we chose to test a CDK inhibitor that has been shown to induceapoptosis in high MYC-expressing cancer cells via synthetic lethality. Wehypothesized that apoptosis would be induced in metastatic cells transitioninginto proliferation, since they appear to upregulate MYC.
After testing this onmice we found that by looking in high resolution at gene expression in singlemetastatic cells, we have uncovered previously unrealized diversity indifferentiation and gene expression relating to the metastatic stage ,anddemonstrate that this approach can facilitate the identification of newpotential drug targets with efficacy against metastatic disease.METHODSTo begin with the analysis the researchers first gathered the cell linesand the xenografts of the tumour tissues, which were grown and acquiredaccording to standard and ethical protocols. The xenografts were divided intotumour fragments and propagated into the breasts of the mice. When the tumoursbecame palpable, that’s when the tumours were measured weekly to oversee theirgrowth rate. The tumour fragments were stored by freezing them in liquidnitrogen. All animals from which xenografts were derived were euthanized at theend, when the tumours had grown to about 20 to 25 mm in size. During theresection experiment, tumours were usually removed when they reached the sizeof about 10 to 12 mm. The animals on which resection was performed were broughtback to their colony and were warranted to grow metastases for 8 weeks, duringwhich lung tissue was gathered and analysed by fluorescence-activated cellsorting (FACS) for human cells.
In order to measure the functional activity of metastatic cells, orthotopictransplant experiments were performed on the animals. Particular metastaticcells in the lymph nodes, as well as particular tumour cells from matchedanimals, were segregated by FACS and combined from various animals. The sortedcells were formed into pellets and inserted into a media. Diluted versions ofthese were inserted into the breasts of 3.5-week-old mice and grafts were takenafter 4.5 months when the primary tumours became 20 mm in size. After this began the dinaciclib treatment experiments, which wereadministered when the tumours became palpable.
The dinaciclib was primed andacquired according to protocol. The mice were randomly appointed to treatmentswhen the tumour cells were transplanted and analysed with the help of thesingle-blind design. In total, 49 animals were injected with the treatmentthree times a week. Animals were measured twice a week to report primary tumourgrowth. The mice were euthanized at the end of the treatment or earlier if thetumour reached 20 mm in diameter.
Animals which developed unfavourable effectswere ruled out of the study. The microarray gene expression values were calculated using some form ofstatistics program. Plasma membrane genes expressed greatly on all of the 15tumour sample xenografts. The 12 initial patient tumour samples were rankedfrom highest to lowest expression. The predicted value of every one of the 55genes characteristics of low-burden metastatic cells was worked out byKaplan–Meier analysis. All solid tissues and the brain were dissociated for FACS. The tissues werecut up and placed in culture medium. They were then broken down for 45 min at37 °C.
The suspensions that arose were then inserted into a solution of DNasefor 3 min at room temperature, after which they were washed and dissociatedagain. After this peripheral blood, supernatant and bone marrow were collected,cells were pelleted for 5 min and leftover erythrocytes in peripheral blood,lung and tumour samples were lysed for 5 min at room temperature. All unusedsamples were directly filtered and stored by freezing them in liquid nitrogen. The tissues from the reduction mammoplasty were washed three to five times,cut into small fragments and digested overnight in a solution. The digested fragmentswere then pelleted for 3 min, frozen and then stored in liquid nitrogen. The antibodies for several particular human antigens were boughtcommercially.
Both human and mouse antibodies were stained. After 15 min oflying on ice, the stained cells were washed to get rid of excess antibodies andput back into the medium. The cells were then flow sorted and analysed. Deadcells were eliminated and contaminating human or mouse haematopoietic andendothelial cells were excluded. The complete tissue sample in the single-cellmultiplex qPCR experiments was run through the flow cytometer. A steady numberof live cells were found in the tissues of all of the animals. The results ofmice which deviated by more than one standard deviation were excluded from thestudy. Single-cell gene-expression experiments were carried out with microfluidchips.
Single cells were sorted using FACS into distinctive wells. Theexperiments were done according to protocol. Each well was prefilled with asolution. After the sorting process, the PCR plated were frozen and or placedinto the thermocycler to go through the process of combined reversetranscription and target-specific amplification.
Exonuclease reaction solutionwas subsequently added to remove unincorporated primers. Each well was thendiluted. A bit from each sample was then dropped into a separate plate andmixed with another solution. Individual primer assay mixes were made in yetanother plate. The chips were primed before the samples and assays were mixedinto them. The chips were then evaluated thoroughly.
All of the single-cell PCR data were analysed using a statistical analysissoftware. In its entirety, 268 mammary cells from reduction mammoplasties aswell as 441 metastatic and 523 primary tumour cells from the xenografts of themice were analysed. The results of the analyses were developed into Ct values,which were then further generated into statistical language. In regular mammary cell experiments, the Ct values were standardized bydeducting the average value of the basal/stem-cell population per gene and perarray. In the mice xenograft experiments, the Ct values were standardized bydeducting the average primary tumour expression per gene as well as the averagevalue of the basal/stem-cell population per gene and per array. Low qualitysamples were found and withdrawn from additional analysis. Various statistical tests were performed in order to determine geneexpression differences between earlier established populations.
For regularmammary cell experiments, a threefold comparison was initiated betweenbasal/stem, luminal, and luminal progenitor cells. This generated an array of49 differentially expressed genes. To find out of which population each gene isan aspect of, pair-wise tests were executed. When comparing metastatic cellexperiments to that of primary tumour cells, only the pair-wise tests wereexecuted. Threefold comparisons were executed to compare lung metastatic cellsfrom the three xenografts from the mice and fivefold comparisons were executedto compare metastatic cells from each tissue.
These analyses were done with avariety of statistical programs. In order to find passages which were represented in a greater fashion inthe set of significantly differentially expressed genes that they would havebeen by just chance, an enrichment analysis of Biological Process gene ontologyterms was executed using several statistical programs. For both histological analysis and immunofluorescent analysis, the tissueswere suspended overnight in paraformaldehyde and processed paraffin embedding.The tissues were stained with haematoxylin and eosin for histological analysis.In order to commence the immunofluorescent analysis, the tissues were stainedusing immunofluorescent staining. The immunofluorescent staining was used uponlung tissues with low and high metastatic burden.On the sections with paraffin-embedded tissue immunostaining was carriedout by using a citrate buffer and heating the sections in a pressure cooker for8 min.
Several human genes were stained with the help of a three-step method.First, the primary antibodies were incubated overnight, which was then followedby one-hour incubations along with detecting antibodies after which afluorescent binding biotin was added. MYC and phospho-histones were found byfollowing a two-step method, in which the overnight staining of antibodies wasfollowed up by a one-hour incubation along with detecting antibodies. Thenumber of positive nuclei was counted for tumour, high burden, and low burden cells,after which the significance was calculated by a statistical analysis software. Metastasis is a process in which cancer cells spread to other sites withinthe body. Metastatic cancer, also known as advanced cancer or stage IV cancer,is defined as the spread of cancer from a primary tumour to the rest of thebody.
This type of cancer is the main cause of fatality (about 90% of deaths)in cancer patients due to the fact that it cannot be fully cured. In order for metastasis to develop the cancer cells must first break awayfrom the primary tumour and travel through the circulatory and lymphaticsystems. Then it must avoid being destroyed by immune attacks initiated by thewhite blood cells and erupt at beds of the capillary kind. After this, the metastasiscan infest into distant organs and proliferate. Metastatic cancer cells alsohave the ability to create a new environment in which harmful secondary tumourscan cultivate. While it is still unknown what exactly the origin is of these metastaticcancer cells there definitely have been multiple hypotheses made about thisexact question. Many hypothesise that metastatic cells emerge during a processin which epithelial cells go through a number of gene mutations whicheventually transforms it into a mesenchymal tumour cell. Other theories statethat metastatic cells stem from several collections of stem cells.
Anothertheory prevails that tumour-associated macrophages are the primary cause ofmetastasis, due to the fact that it has the ability to set up a pre-metastaticalcove and encourages tumour inflammation. The last theory states thatmetastatic cancer cells appear due to inability to take up enough oxygen and/orexpel enough carbon dioxide in myeloid cells. Metastatic cancer doesn’t just spread sporadically, instead, depending onthe type cancer, it will spread to specific sites in the body. For example,with breast cancer the metastasis has the urge to spread mostly to the liver,lungs, bones, and brains, while pancreas cancer has the tendency to spread tothe peritoneum, lungs and liver. While metastatic cannot be cured yet, it can be treated in order to prolongthe life of the patient and improve their wellbeing.
Typically this treatmentaims at slowing the growth of the metastasis and relieving the symptoms thepatient may experience due to the metastasis. The type of treatment depends onthe type of cancer, the size of the metastasis, where the metastasis started,the location of the metastasis and several other factors that may intervenewith the condition of the patient or the metastasis. Treatment for metastatic cancer typically includes systemic therapy ortherapeutics which are ingested or injected into the bloodstream. The mostcommon form of these therapeutics is chemotherapy or hormone therapy. Othertreatments to reduce the effects of metastasis are treatments such as radiationtherapy, biological therapy and/or surgery. MetastaticBreast Cancer to the Brain: A Clinical Primer for Translational InvestigationWhiletreatment for primary breast cancer becomes more and more effective there is alack of clinical advancement in the area of brain metastasis from breastcancer. In order to broaden the spectrum of brain metastasis treatments, wemust first focus on the subtype of breast cancer, the evolution of breastcancer subtypes as metastases form, and the general medical condition of thepatient.
Treatmentsfor brain metastasis are currently scarce and not effective enough with amaximum life expectancy of 16 months after the start of the treatment. Whilesurgically removing any lesions will definitely improve the patient’scondition, there is a large chance of tumour recurrence unless the surgery isfollowed up with radiotherapy. While radiotherapy is successful in targetingand removing cancer cells throughout the whole brain, there have been reportsof significant cognitive decline due to the exposure of radiation to the braintissue. Lastly, while medical therapeutics may have the ability to rid ofcancer cells with little to no harmful side effects many chemotherapeuticagents are unable to pass the blood brain barrier and reach the tumour. In conclusion, in order for brain metastasis to be moresuccessfully treated we must focus on discovering new therapeutics that cancross the blood brain barrier and target tumour cells.
Jammed Cells Expose the Physics of CancerIn 1995 PeterFriedl had a startling discovery: coordinated cells which the had been growingin his lab started clustering together and started moving through a network offibers which were meant to mimic the human body.His discoverywas important for the research about jamming, a process where different cellspack together so tightly that they become one unit. Cells in a tumor or tissuecan change their own mechanical properties because of their mechanicalmicroenvironment, using genetic programs and other feedback loops, and ifjamming is to provide a solid conceptual foundation for aspects of cancer, itwill need to account for this ability. Theories forhow cancer might behave mechanically have only been researched as theories forsolids or theories for fluids, however a small group of scientists havesuspected that a combination of sticky epithelial cells, which make up the bulkof solid tumors, and thinner, more mobile mesenchymal cells that are oftenfound circulating solo in cancer patients’ bloodstreams could cause a phasetransition . Scientists also found that a phase transition between jammed andunjammed states could fluidize and mobilize tumor cells as a group withoutrequiring them to transform from one cell type to a drastically different one.