Inflammation and Cancer: Methods and Protocols: Volume 2: Molecular Analysis and Pathways
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Our Day return guarantee still applies. Advanced Book Search Browse by Subject. Make an Offer. To demonstrate our hypothesis, we first immunophenotyped the SNOs exploiting their high expression of N-cadherin. SNOs were also less proliferating Fig. Ki staining further confirmed an SNO-mediated impairment of cancer cell proliferation Fig.
Yellow arrowhead, mammosphere. They were stained with red PKH26 and expected to lose this stable membrane dye during cytokinesis Supplementary Fig. The high expression of Jagged1 in SNOs prompted us to investigate the relevance of the Jagged1 partner proteins, Notch, in the mechanism of breast cancer cellular dormancy. Single-cell analysis using confocal imaging was performed for all the Notch proteins and revealed that they were more highly expressed on the surface of MDA GFP cells bound to SNOs, with the strongest expression observed for Notch2 Fig.
This analysis demonstrated that silencing Notch2 and Notch1 reversed the SNO-mediated inhibitory effect on proliferation, with the Notch2 downregulation effect resulting more potent than the downregulation of Notch1. These results prompted us to narrow down our investigation to the Notch2 pathway. Taken together, these results suggest that dormant MDA-MB cells mimic HSCs not only by using endosteal niche signals for quiescence but also maintaining a stem phenotype, possibly indispensable to initiate new tumours after reactivation. Of note, despite their lower proliferation in standard 2D cultures Supplementary Fig.
However, owing to the potent aggressiveness of 4T1 cells, all mice developed osteolytic lesions within 15 days of cell injection Supplementary Fig. Nevertheless, we observed that single cytokeratin-positive 4T1 cells lying near the N-cadherin- positive endosteal osteoblasts Supplementary Fig.
Importantly, these tumour cells were negative for the proliferation markers Ki Fig. Of note, these tumour cells were also N-cadherin positive Fig. Notch2 HIGH breast cancer cells. Since resistance to chemotherapy is a hallmark of dormant cancer cells, we tested whether single MDA-MB cells were still lodging in the endosteal area after treatment with doxorubicin Supplementary Fig. The treatment was effective in reducing the incidence Supplementary Fig. In contrast, histological analysis of bones revealed a higher number of cancer cells in Notch2 LOW cell-injected mice Fig.
To better investigate the cause of weight loss in MDA-Notch2 LOW cell-injected mice, which is normally unexpected when tumour cells are directly injected into the tibia medullary cavity, we examined anatomically the visceral organs, observing no macroscopic metastases. However, histological analysis of liver sections demonstrated that tumour cells spread to this organ forming microscopic multicellular metastases.
Altogether, these results confirmed both the relevance of the Notch2 pathway in the cellular dormancy and the role of the bone in the metastatic dissemination of breast cancers to vital visceral organs. To address the relevance of the Notch pathway in the in vivo breast cancer cellular dormancy and subsequent mobilisation, we tested whether acute Notch inhibition could reactivate the dormant breast cancer cells in our model of dormancy.
After 4 weeks of in vivo dormancy intratibial injection of MDA LUC cells and absence of osteolytic lesions and bioluminescent signal , mice were randomised and divided into two groups. One group received a single acute injection of 4. Notch inhibition.
After 42 days from cell injection, tumour-free mice negative to osteolysis and luciferase bioluminescence were assumed to harbour dormant cells and were included in the study. They were randomly assorted into two groups receiving a single injection of vehicle dimethyl sulfoxide or of 4. Nuclear counterstain, DAPI blue ; red arrows, distance from endosteum.
Acquisition of the microarray data
In the timeframe of 2 months after the DBZ treatment, we did not observe overt tumours, neither in the bone nor in the visceral organs Supplementary Fig. However, histomorphometric evaluation of the tibias showed that the breast cancer cell distance from the bone was significantly increased by the single DBZ treatment Fig.
Consequently, the number of breast cancer cells nearby the endosteal niche decreased Fig. Tumour cell distribution analysis confirmed that the acute Notch inhibition significantly altered the in vivo distribution of breast cancer cells in the bone marrow, inducing their mobilisation from the endosteal niche Fig. Notably, the histological analysis of distal colonisation of the liver revealed that the number of detectable multicellular metastases, as well as their size, were increased by the acute DBZ treatment Fig.
To examine whether chronic Notch inhibition could instead stimulate the local occurrence of osteolytic lesions, a similar experiment was conducted treating mice with 4. Even with this chronic experimental setting, no osteolytic lesions appeared in DBZ-treated mice Supplementary Fig. With the aim to broaden our findings and identify common pathological conditions that could potentially reactivate dormant breast cancer cells, we investigated the effects on dormancy of acute and chronic inflammation.
After 4 weeks of dormancy intratibial injection of MDA LUC cells and absence of osteolysis and bioluminescence signal , we treated mice with a single injection of vehicle PBS or 1.
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Tumour cell distribution analysis revealed that the acute inflammation failed to alter the in vivo territorial lodging of MDA CYTK in the bone marrow Fig. Analysis of distal colonisation revealed that the number of liver metastases, as well as their size, was not changed by the treatment Fig. Role of inflammation. Mice with dormancy were randomly divided into two groups and treated with a single injection of 1.
Subsequently, we tested whether chronic pro-inflammatory treatment could reactivate cells after 4 weeks of dormancy. Mice with breast cancer dormancy were randomised and divided into two groups, each receiving a daily injection of vehicle PBS or 0. In the timeframe of 2 months of chronic LPS treatment, we did not observe overt tumours, neither in the bone nor in the visceral organs. In this model, histomorphometric evaluation of MDA CYTK distance from the bone also did not show significant differences between the two treatments Fig. Tumour cell distribution analysis revealed that the chronic inflammation also did not alter the in vivo distribution of MDA CYTK in the bone marrow Fig.
Distal colonisation revealed that the number of liver metastases was not changed by the treatment Fig. Taken together, these results ruled out any effect of acute or chronic inflammation in the mobilisation of dormant breast cancer cells and initiation of new cancers, albeit confirming the known pro-tumour role of chronic inflammation. Using the SurvExpress software, 31 we enquired 23 data sets representing breast cancers from patients.
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Total population analysis showed a positive statistical correlation between high Notch2 levels and overall survival Fig. Moreover, when we stratified patients according to the treatment, we found a positive correlation of Notch2 HIGH expression and survival in untreated patients Fig. Intriguingly, Notch2 HIGH was no longer correlated with a better prognosis under the selective pressure of chemotherapy Fig. However, it is important to note that the divergence of the curves appears in the first 5 years, therefore we cannot exclude that they were not also due to a different rate of tumour proliferation.
Furthermore, when data were stratified for oestrogen receptor subtypes, we confirmed a significant and positive correlation between high Notch2 levels and overall survival in untreated patients with either oestrogen receptor-positive or -negative breast cancers Fig. Notch2 expression in human primary tumours and correlation with survival. Representative immunohistochemistry for Notch2 in mated primary tumours and i associated bone metastasis. Statistical analyses: a Z -test, b — g log-rank test.
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Finally, we investigated the expression of Notch2 in samples from a cohort of bone metastatic breast cancer patients both in primary tumours and matched bone metastases. This study provides a new understanding of breast cancer cellular dormancy and mobilisation processes in the bone microenvironment.
Dormant tumour cells compete with HSCs for bone marrow engraftment and lodge close to the endosteal niche made up by N-cadherin-positive osteoblasts, also known as SNOs. Disruption of this pathway reactivates dormant cells and appears to mobilise them, making their distance from SNOs significantly higher and inducing greater numbers of metastatic foci in the liver, likely through bone marrow tumour cell escaping into the circulation.
One limitation of the study was that we were unable to demonstrate whether the lower body weight was caused by the metastatic spreading of Notch2 LOW cells, leaving this question unanswered. Intriguingly, the role of the Notch pathway in tumour biology is very complex. Data in literature demonstrated that Notch1 and Notch3 are pro-oncogenic, 33 , 34 , 35 and recently it has been shown that gain of Notch2 copy number is associated with poor prognosis in metastatic triple-negative breast cancer.
Furthermore, Notch and its receptors have been shown to be involved in heterotypic interactions triggering escape from tumour dormancy in a model of angiogenesis-dependent cancer dormancy. This leads to the conclusion that Notch has multifaceted roles in tumour biology and that the positive or negative outcomes are likely to depend on a fine tuning of their balance. According to our results, the endosteal niche could be considered the place where tumour cell dormancy takes place, followed by mobilisation to initiate a new tumour when the dormancy signals are disrupted. Therefore, tumour dormancy and initiation can be considered two sides of the same coin as the long-term initiation of a new tumour is the pathologic manifestation that dormancy occurred previously.
Furthermore, dormant cancer cells share with HSCs the expression of stem genes e. Furthermore, like HSCs, dormant cancer cells express a drug-resistant phenotype, which contributes to their survival when cancers are challenged by chemotherapy. Given the complexity of the concept, more work will be necessary to dissect the underlying mechanisms in detail. Notch2 HIGH cancer cells are also observed in human breast carcinomas.
They are already detectable in primary tumours, where they represent a small portion of the total cancer cell population. In these samples, we do not have dynamic observations that can predict the occurrence of cellular dormancy, which represent another limitation of the study. However, analysis of public data sets showed a better survival in patients bearing breast cancers expressing higher levels of Notch2, which was even more prominent in untreated patients or in patients treated with endocrine therapy.
In contrast, the fact that this advantage is lost in patients after chemotherapy, suggests that drug-resistance may also be a hallmark of Notch2 HIGH cells in human breast cancers and that this selection can make the relapse more likely if any environmental condition mobilises and reactivates the dormant cells. Our experimental setting certainly correlated the disruption of the Notch signal with dormant cell mobilisation and colonisation of the liver.
Interestingly, upon acute Notch signal disruption by DBZ, cancer cells appear unable to relocate near the endosteum, suggesting a persistent molecular change, perhaps also induced by signals emanated by the microenvironmental compartment apart from the endosteal surface. In contrast, both acute and chronic inflammation appear not to play a role in dormancy. However, in our experimental conditions, chronic inflammation increased the number of cancer cells in the bone marrow and the size of liver metastases, consistent with reports demonstrating the role of inflammation in cancer progression, 44 but this effect seems not to be exerted on the Notch2 HIGH cells resident near the endosteal niche.