Dormant Phenotype Research Articles

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood.In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination.In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.

Genetically engineered control of phenotypic structure in microbial colonies.

Rapid advances in cellular engineering1,2 have positioned synthetic biology to address therapeutic3,4 and industrial5 problems, but a substantial obstacle is the myriad of unanticipated cellular responses in heterogeneous real-world environments such as the gut6,7, solid tumours8,9, bioreactors10 or soil11. Complex interactions between the environment and cells often arise through non-uniform nutrient availability, which generates bidirectional coupling as cells both adjust to and modify their local environment through phenotypic differentiation12,13. Although synthetic spatial gene expression patterns14-17 have been explored under homogeneous conditions, the mutual interaction of gene circuits, growth phenotype and the environment remains a challenge. Here, we design gene circuits that sense and control phenotypic structure in microcolonies containing both growing and dormant bacteria. We implement structure modulation by coupling different downstream modules to a tunable sensor that leverages Escherichia coli's stress response and is activated on growth arrest. One is an actuator module that slows growth and thereby alters nutrient gradients. Environmental feedback in this circuit generates robust cycling between growth and dormancy in the interior of the colony, as predicted by a spatiotemporal computational model. We also use the sensor to drive an inducible gating module for selective gene expression in non-dividing cells, which allows us to radically alter population structure by eliminating the dormant phenotype with a 'stress-gated lysis circuit'. Our results establish a strategy to leverage and control microbial colony structure for synthetic biology applications in complex environments.

A new in vitro dormancy model of <i>Mycobacterium tuberculosis</i>

The article describes a new in vitro dormancy model of M. tuberculosis based on cultivation with deprivation of four components of the standard medium of Mediumbrook 7H9. The modeling method and characteristics of the obtained dormant phenotype of M. tuberculosis are described in detail, and fields for potential application of the model are proposed.

Mesenchymal Stem/Stromal Cell Engulfment by Breast Cancer Cells Generates a Hybrid Cancer Cell Population with Dormancy and Chemoresistance

IntroductionDrug‐resistant dormant tumor cells are a major causes of breast cancer relapse and metastasis. It has been reported that in the tumor microenvironment breast cancer cells (BCC) can engulf mesenchymal stem/stromal cells (MSCs) and acquire dormancy. We previously documented the presence of BCC that engulf MSCs in clinical samples of breast cancer metastasis and showed that MSC engulfment leads to a more aggressive breast cancer phenotype, with an upregulated gene signature consisting of MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1. However, the mechanisms of MSC engulfment, and the phenotypic features of hybrid breast cancer cells are still unclear. Here, we tested the hypothesis that MSC engulfment generates hybrid cancer cells exhibit a multinucleated phenotype and increased survival advantage, dormancy, and chemoresistance, and that the genes we identified may mediate MSC engulfment.MethodMSC labeled with DS‐RED and MDA‐MB‐231 labeled with GFP (GFP‐231) were co‐cultured in microfluidic cell pairing devices, and treated with antibodies against MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1 or IgG control to quantify the percentage of MSC engulfing on‐chip. The conditioned media was subjected to a cytokine array. Live Imaging Microscopy was used to characterize the hybrid cancer cells in co‐cultures compared the GFP‐231. In Co‐cultures of DS‐RED‐MSC with GFP‐231 stably transduced with shWNT5A, shMSR1 or lentivirus control, and treated with Doxorubicin or untreated, we analyzed the percentage of DS‐RED+/GFP+ hybrid population, Ki‐67 low cells in G1, multinucleated cells (N4+), and the percentage of apoptotic cells using annexin V/PI.ResultsNeutralizing antibodies or shRNAs against WNT5A and MSR1 significantly reduced the percentage of MSC engulfment by MDA‐MB‐231 cells, and changed the cytokine profile in the conditioned medium of co‐cultures. In co‐cultures with DS‐RED‐MSCs, DS‐RED+/GFP+ hybrid cells had a higher percentage of Ki‐67 low in G1 and higher percentage of aneuploidy compared to GFP‐231+ cells, which were reversed by WNT5A and MSR1 shRNA downregulation. Doxorubicin treatment increased the percentage of DS‐RED+/GFP+ hybrids cells and decreased the percentage of apoptosis in the co‐cultures when compared to untreated controls.ConclusionsMSC engulfment by BCCs results in a hybrid multinucleated cell population with increased percentage of aneuploidy than BCC controls and a dormant phenotype characterized by higher of Ki‐67 low in G1 compared to controls. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of pro‐metastatic proteins including thrombosponding‐1, UPAR, and VEGF. Hybrid cancer cells show increased resistance to Doxorubicin compared to control BCCs, which can be rescued by downregulation of WNT5A and MRS1.Support or Funding InformationRole of EZH2 Breast Cancer Progression – R01 to C. Kleer. Role of CCN6 (WISP3) in the progression and metastasis – R01 to C. Kleer‐ AIRC Investigator Grant (IG#18602) to S. Andò‐PRIN2015#2015B7M39T to S. Andò

In vitro Models of Breast Cancer Metastatic Dormancy.

Delayed relapses at distant sites are a common clinical observation for certain types of cancers after removal of primary tumor, such as breast and prostate cancer. This evidence has been explained by postulating a long period during which disseminated cancer cells (DCCs) survive in a foreign environment without developing into overt metastasis. Because of the asymptomatic nature of this phenomenon, isolation, and analysis of disseminated dormant cancer cells from clinically disease-free patients is ethically and technically highly problematic and currently these data are largely limited to the bone marrow. That said, detecting, profiling and treating indolent metastatic lesions before the onset of relapse is the imperative. To overcome this major limitation many laboratories developed in vitro models of the metastatic niche for different organs and different types of cancers. In this review we focus specifically on in vitro models designed to study metastatic dormancy of breast cancer cells (BCCs). We provide an overview of the BCCs employed in the different organotypic systems and address the components of the metastatic microenvironment that have been shown to impact on the dormant phenotype: tissue architecture, stromal cells, biochemical environment, oxygen levels, cell density. A brief description of the organ-specific in vitro models for bone, liver, and lung is provided. Finally, we discuss the strategies employed so far for the validation of the different systems.

An in vitro hyaluronic acid hydrogel based platform to model dormancy in brain metastatic breast cancer cells

Breast cancer cells (BCCs) can remain dormant at the metastatic site, which when revoked leads to formation of metastasis several years after the treatment of primary tumor. Particularly, awakening of dormant BCCs in the brain results in breast cancer brain metastasis (BCBrM) which marks the most advanced stage of the disease with a median survival period of ~4–16 months. However, our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. To address this need, we developed an in vitro hyaluronic acid (HA) hydrogel platform to model dormancy in brain metastatic BCCs via exploiting the bio-physical cues provided by HA hydrogels while bracketing the normal brain and metastatic brain malignancy relevant stiffness range. In this system, we observed that MDA-MB-231Br and BT474Br3 brain metastatic BCCs exhibited a dormant phenotype when cultured on soft (0.4 kPa) HA hydrogel compared to stiff (4.5 kPa) HA hydrogel as characterized by significantly lower EdU and Ki67 positivity. Further, we demonstrated the nuclear localization of p21 and p27 (markers associated with dormancy) in dormant MDA-MB-231Br cells contrary to their cytoplasmic localization in the proliferative population. We also demonstrated that the stiffness-based dormancy in MDA-MB-231Br cells was reversible and was, in part, mediated by focal adhesion kinases and the initial cell seeding density. Finally, RNA sequencing confirmed the dormant phenotype in MDA-MB-231Br cells. This platform could further our understanding of dormancy in BCBrM and could be adapted for anti-metastatic drug screening. Statement of SignificanceOur understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. Herein, we present a HA hydrogel-based platform to model dormancy in brain metastatic BCCs while recapitulating key aspects of brain microenvironment. We demonstrated that the biophysical cues provided the HA hydrogel mediates dormancy in brain metastatic BCCs by assessing both proliferation and cell cycle arrest markers. We also established the role of focal adhesion kinases and initial cell seeding density in the stiffness-mediated dormancy in brain metastatic BCCs. Further, RNA-seq. confirmed the dormant phenotype in brain metastatic BCCs. This platform could be utilized to further our understanding of microenvironmental regulation of dormancy in BCBrM.

Dormant tumor cells interact with memory CD8+ T cells in RET transgenic mouse melanoma model.

Melanoma is an aggressive form of skin-cancer. Melanoma cells are characterized by their plasticity, resulting in therapy resistance. Using RET transgenic mouse melanoma model, we characterized dormant tumor cells accumulated in the bone marrow (BM) and investigated their interaction with effector memory CD8+ T cells. We found that cells expressing melanoma-associated antigen tyrosinase related protein (TRP)-2 and stemness marker CD133 represented less than 1.5% of all melanoma cells in primary skin lesions and metastatic lymph nodes. The majority of these cells were negative for the proliferation marker Ki67. In the BM, CD133+TRP-2+ melanoma cells displayed an aberrant expression of p16, p27, Ki67 and PCNA proteins, suggesting their dormant phenotype. Moreover, these cells were characterized by an elevated expression of various molecules characterized stemness, metastatic, angiogenic and immunosuppressive properties such as CD271, CD34, HIF-1α, CXCR3, CXCR4, VEGR2, PD-L1, CTLA-4, CD39 and CCR4 as compared to their CD133- counterparts. Disseminated BM dormant TRP-2+ tumor cells were found to be co-localized with memory CD8+ T cells. Our data suggest that these dormant melanoma cells in the BM could play an important role in the maintenance of memory T cells in the BM.

Matrix stiffness and cluster size collectively regulate dormancy versus proliferation in brain metastatic breast cancer cell clusters.

Breast cancer cells can metastasize either as single cells or as clusters to distant organs from the primary tumor site. Cell clusters have been shown to possess higher metastatic potential compared to single cells. The organ microenvironment is critical in regulating the ultimate phenotype, specifically, the dormant versus proliferative phenotypes, of these clusters. In the context of breast cancer brain metastasis (BCBM), tumor cell cluster-organ microenvironment interactions are not well understood, in part, due to the lack of suitable biomimetic in vitro models. To address this need, herein, we report a biomaterial-based model, utilizing hyaluronic acid (HA) hydrogels with varying stiffnesses to mimic the brain microenvironment. Cell spheroids were used to mimic cell clusters. Using 100-10 000 MDA-MB-231Br BCBM cells, six different sizes of cell spheroids were prepared to study the impact of cluster size on dormancy. On soft HA hydrogels (∼0.4 kPa), irrespective of spheroid size, all cell spheroids attained a dormant phenotype, whereas on stiff HA hydrogels (∼4.5 kPa), size dependent switch between the dormant and proliferative phenotypes was noted (i.e., proliferative phenotype ≥5000 cell clusters < dormant phenotype), as tested via EdU and Ki67 staining. Furthermore, we demonstrated that the matrix stiffness driven dormancy was reversible. Such biomaterial systems provide useful tools to probe cell cluster-matrix interactions in BCBM.

CBMT-27. ABERRANT AMINO ACID METABOLISM EXPOSE NOVEL FUNCTIONAL VULNERABILITIES IN GLIOBLASTOMA

Abstract Despite advances in molecularly characterizing glioblastoma, metabolic alterations driving its aggressive phenotype are only beginning to be recognized. Integrative cross-platform analyses coupling global metabolomic and gene expression profiling identified aberrant amino acid (AA) metabolism as a central node in glioblastoma. This metabolic phenotype was recapitulated in preclinical models and through a series of investigations designed to determine the biologic consequence of individual AA, we identified branched chain AA (BCAA) and glutamine as the only indispensable AA in glioblastoma, serving as the sole source of nucleotide pools and glutathione, respectively. Although molecularly and/or chemically perturbing these pathways resulted in cytotoxicity in glioblastoma, normal astrocytes demonstrated a similar response, suggesting therapeutic limitations in targeting these core metabolic pathways in cancer. As the glioblastoma microenvironment typically represents a nutrient-deprived state, we went on to determine the capacity of these cells to adapt to AA restricted conditions by only providing these cells with the above-identified indispensable AA. Intriguingly, glioblastoma cells had the unique ability to revert a state of metabolic dormancy. In addition to triggering a reversible proliferative arrest, this dormant phenotype displayed a near-complete shutdown of glycolysis that allowed these cells to adapt and maintain survival in glucose-deprived conditions and elicited profound resistance to ionizing radiation. Studies designed to systemically understand molecular underpinnings driving this unique metabolically dormant state uncovered a functional reliance upon mTOR/p21 signaling to maintain proliferative arrest in nutrient unfavorable conditions. Consistent with these findings, p21 expression was differentially expressed in the perinecrotic core of glioblastoma when compared to the peripheral edge in patient samples. Targeting this novel functional vulnerability through p21 inhibition, thereby, ‘forcing’ proliferation of these cells in nutrient unfavorable conditions, led to robust cytotoxicity specific to dormant cells and enhanced radiation response. Targeting functional vulnerabilities in otherwise therapeutically resistant cells represents a promising clinical strategy in glioblastoma.

Using diphenyleneiodonium to induce a viable but non-culturable phenotype in Mycobacterium tuberculosis and its metabolomics analysis.

Depletion of oxygen levels is a well-accepted model for induction of non-replicating, persistent states in mycobacteria. Increasing the stress levels in mycobacterium bacilli facilitates their entry into a non-cultivable, dormant state. In this study, it was shown that diphenyleneiodonium, an inhibitor of NADH oxidase, induced a viable, but non-culturable state in mycobacteria, having similar features to dormant bacilli, like loss of acid-fastness, upregulation of stress-regulated genes and decreased superoxide levels as compared to actively growing bacilli. Comprehensive, untargeted metabolic profiling also confirmed a decrease in biogenesis of amino acids, NAD, unsaturated fatty acids and nucleotides. Additionally, an increase in the level of lactate, fumarate, succinate and pentose phosphate pathways along with increased mycothiol and sulfate metabolites, similar to dormant bacilli, was observed in the granuloma. These non-cultivable bacilli were resuscitated by supplementation of fetal bovine serum, regaining their culturability in liquid as well as on agar medium. This study focused on the effect of diphenyleneiodonium treatment in causing mycobacteria to rapidly transition from an active state into a viable, but non-cultivable state, and comparing their characteristics with dormant phenotypes.

Escherichia coli K12: An evolving opportunistic commensal gut microbe distorts barrier integrity in human intestinal cells

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.

Freezing Tolerance of Thermophilic Bacterial Endospores in Marine Sediments.

Dormant endospores of anaerobic, thermophilic bacteria found in cold marine sediments offer a useful model for studying microbial biogeography, dispersal, and survival. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to be isolated and studied independently of other factors such as environmental selection. To study the resilience of thermospores to conditions relevant for survival in extreme cold conditions, their viability following different freezing treatments was tested. Marine sediment was frozen at either −80°C or −20°C for 10 days prior to pasteurization and incubation at +50°C for 21 days to assess thermospore viability. Sulfate reduction commenced at +50°C following both freezing pretreatments indicating persistence of thermophilic endospores of sulfate-reducing bacteria. The onset of sulfate reduction at +50°C was delayed in −80°C pretreated microcosms, which exhibited more variability between triplicates, compared to −20°C pretreated microcosms and parallel controls that were not frozen in advance. Microbial communities were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative sequence abundance of thermophilic endospore-forming Firmicutes in all microcosms. Different freezing pretreatments (−80°C and −20°C) did not appreciably influence the shift in overall bacterial community composition that occurred during the +50°C incubations. Communities that had been frozen prior to +50°C incubation showed an increase in the relative sequence abundance of operational taxonomic units (OTUs) affiliated with the class Bacilli, relative to unfrozen controls. These results show that freezing impacts but does not obliterate thermospore populations and their ability to germinate and grow under appropriate conditions. Indeed the majority of the thermospore OTUs detected in this study (21 of 22) could be observed following one or both freezing treatments. These results are important for assessing thermospore viability in frozen samples and following cold exposure such as the very low temperatures that would be encountered during panspermia.

Ecobiological approach to research regarding ageing and diseases.

Ageing and age-related diseases (ARD) share a common biological clock that appears as their root cause: protein damage. A majority of proteins have evolved into native structures resistant to oxidative damage but any folding imperfections, including those due to "silent" amino acid substitutions, reduce oxidation resistance. Damaged proteins accumulate with age and trigger ageing-like phenotypes reversible by their turnover, while acquired genome alterations remain as stable consequences of protein malfunction. Ageing and ARD display species-specific latency in phenotypic expression. Disease latency may be proposed as to be due to phenotypic suppression of cellular defects by molecular traffic among neighbouring cells. Such cross-complementation of functional deficiencies acts as a kind of tissue-based cellular "solidarity", called cellular parabiosis. Chronic inflammation reveals dormant cell phenotypes and shortens disease latency by the breakdown of cell-cell communication, as in tumour promotion and inflammation. At the present time, predictive diagnostics, prognostics, prevention and even cure of disease by phenotypic reversion become conceivable.

Tumor Cell Dormancy and Reactivation in Bone: Skeletal Biology and Therapeutic Opportunities.

ABSTRACTIn the advanced stages of many cancers, tumor cells disseminate from the primary site and colonize distant locations such as the skeleton. These disseminated tumor cells colonizing bone can evade treatments and survive for prolonged periods in a dormant state before becoming reactivated to form overt metastases. The precise interactions between tumor cells and the bone microenvironment that promote survival, dormancy, and reactivation are currently unknown; as a result, bone metastases remain incurable. In this review we discuss the unique cellular and microenvironmental features of endosteal bone that tumor cells engage with to persist and survive, and ultimately reactivate and proliferate. Specifically, we provide a detailed summary of current perspectives on the processes of tumor cell colonization of the skeleton, and the endosteal bone cells as critical controllers of the dormant cancer cell phenotype, as well as relevant microenvironmental effects such as hypoxia. Evidence for the role of the osteoclast in controlling dormant cancer cell reactivation in bone is highlighted, preceding a discussion of therapeutics targeting the bone microenvironment, including anti‐RANK ligand and bisphosphonate therapies and their potential utility in preventing tumor cell reactivation in addition to protecting bone from tumor‐induced destruction. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER−) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER− breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4+/CD8+ T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER− breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER− breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER− breast cancer patients treated with (neo)adjuvant chemotherapy.

Long-Term Dormant Cells in Acute Myeloid Leukemia Patient-Derived Xenografts Display Reversible Treatment Resistance, but Are Not Enriched for Leukemia-Initiating Cells

IntroductionLeukemia-initiating cells (LIC) are considered the source of therapy resistance and relapse, including in acute myeloid leukemia (AML). Resistance of LIC to standard chemotherapy is often attributed to the quiescent phenotype of LIC. Whether biological characteristics relevant for therapy resistance are stable or reversible traits of LIC is unknown.AimHere, we asked whether long-term dormant cells might exist in AML patient-derived xenografts (PDX) and which characteristics they might have.MethodsWe analyzed 8 serially transplantable PDX models, originally established from AML patients of various groups. We genetically engineered AML PDX cells to express luciferase and a red fluorophore to enable in vivo imaging and isolation of very low numbers of human cells from mice. To track proliferation dynamics of individual cells, we labeled PDX with the proliferation sensitive dye CFSE. Cells were re-isolated from murine bone marrow at different time points after transplantation, and CFSE content, which is reduced in bisections by every cell division, was analyzed by flow cytometry. Long-term dormant cells were defined as cells that had undergone less than 3 cell divisions, while rapidly growing cells had divided more than 7 times during 10 days of in vivo growth.ResultsA rare subpopulation of long-term dormant cells was detected at day 14 in all but one pediatric AML PDX samples, despite a general increase in tumor load by several orders of magnitude. Time course analysis of 3 samples for up to 29 days revealed that dormant tumor cells can still be detected at later time points. These data indicate that the majority of AML PDX samples contain a rare fraction of long-term dormant tumor cells.As dormancy is often attributed to cancer stem cells, we tested whether LIC are enriched in the long-term dormant compartment. Dormant and cycling cells were isolated from the murine bone marrow following 10 days of in vivo growth and LIC frequency analyzed by limiting dilution transplantation assay. Interestingly, although only dormant cells display the typical stem cell characteristic of dormancy, both dormant and cycling cells exhibited similar LIC frequency in both samples tested. This indicates that not the entire LIC pool is dormant in AML, but that AML LIC can reside either in a dormant or a proliferating state.To determine whether dormancy is a constant or reversible trait of the dormant subpopulation, we first asked whether dormant cells can re-induce a tumor with similar growth kinetics as cycling cells upon re-transplantation. Re-transplantation of low numbers of dormant or cycling cells revealed that all tumors showed identical growth behaviour, indicating that dormant cells are capable to switch into proliferation after re-transplantation. Second, we asked if proliferating cells are able to switch into dormancy. Therefore, high cell numbers of bulk cells and cycling cells from 1st recipient mice were re-labeled with CFSE, transplanted into secondary recipient mice, and CFSE distribution was analyzed after 10 or 15 days. Transplanted cycling cells and bulk cells gave rise to a similar fraction of dormant cells, indicating that cycling cells are capable to switch into dormancy after re-transplantation. These data reveal a remarkable functional plasticity of AML PDX cells in mice.To determine whether dormant and cycling cells differ in their drug response, mice were injected with CFSE labeled PDX cells and treated from day 7 to 9 with Cytarabine and with DaunoXome on day 7. Treatment response was analyzed at day 10. Treatment reduced tumor burden by approximately one order of magnitude compared to control mice in all 4 PDX samples analyzed. Importantly, the relative proportion of dormant cells within all re-isolated AML cells increased after treatment. This data indicates that dormant cells preferentially survive chemotherapy.ConclusionIn summary, our data demonstrate that AML PDX samples contain a rare subpopulation of dormant and treatment resistant cells. We show that dormancy is a reversible trait of AML PDX. As dormant and cycling cells contain similar LIC frequencies, our data provide evidence that dormancy and stemness are independent traits of AML. In conclusion, dormancy as well as the reversibility of the dormant phenotype are important biological characteristics of AML that need to be considered when designing treatment strategies that aim to eradicate drug resistant LIC. DisclosuresNo relevant conflicts of interest to declare.

Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells

BackgroundAdhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes.MethodsQuantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1’s association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition.ResultsLower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1.ConclusionOverall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

BackgroundDormant estrogen receptor positive (ER+) breast cancer micrometastases in the bone marrow survive adjuvant chemotherapy and recur stochastically for more than 20 years. We hypothesized that inflammatory cytokines produced by stromal injury can re-awaken dormant breast cancer cells.MethodsWe used an established in vitro dormancy model of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells incubated at clonogenic density on fibronectin-coated plates to determine the effects of inflammatory cytokines on reactivation of dormant ER+ breast cancer cells. We measured induction of a mesenchymal phenotype, motility and the capacity to re-enter dormancy. We induced secretory senescence in murine stromal monolayers by oxidation, hypoxia and estrogen deprivation with hydrogen peroxide (H2O2), carbonyl-cyanide m-chlorophenylhydrazzone (CCCP) and Fulvestrant (ICI 182780), respectively, and determined the effects on growth of co-cultivated breast cancer cells.ResultsExogenous recombinant human (rh) interleukin (IL)-6, IL-8 or transforming growth factor β1 (TGFβ1) induced regrowth of dormant MCF-7 cells on fibronectin-coated plates. Dormant cells had decreased expression of E-cadherin and estrogen receptor α (ERα) and increased expression of N-cadherin and SNAI2 (SLUG). Cytokine or TGFβ1 treatment of dormant clones induced formation of growing clones, a mesenchymal appearance, increased motility and an impaired capacity to re-enter dormancy. Stromal injury induced secretion of IL-6, IL-8, upregulated tumor necrosis factor alpha (TNFα), activated TGFβ and stimulated the growth of co-cultivated MCF-7 cells. MCF-7 cells induced secretion of IL-6 and IL-8 by stroma in co-culture.ConclusionsDormant ER+ breast cancer cells have activated epithelial mesenchymal transition (EMT) gene expression programs and downregulated ERα but maintain a dormant epithelial phenotype. Stromal inflammation reactivates these cells, induces growth and a mesenchymal phenotype. Reactivated, growing cells have an impaired ability to re-enter dormancy. In turn, breast cancer cells co-cultured with stroma induce secretion of IL-6 and IL-8 by the stroma, creating a positive feedback loop.

A dormant TIL phenotype defines non-small cell lung carcinomas sensitive to immune checkpoint blockers

The biological determinants of sensitivity and resistance to immune checkpoint blockers are not completely understood. To elucidate the role of intratumoral T-cells and their association with the tumor genomic landscape, we perform paired whole exome DNA sequencing and multiplexed quantitative immunofluorescence (QIF) in pre-treatment samples from non-small cell lung carcinoma (NSCLC) patients treated with PD-1 axis blockers. QIF is used to simultaneously measure the level of CD3+ tumor infiltrating lymphocytes (TILs), in situ T-cell proliferation (Ki-67 in CD3) and effector capacity (Granzyme-B in CD3). Elevated mutational load, candidate class-I neoantigens or intratumoral CD3 signal are significantly associated with favorable response to therapy. Additionally, a “dormant” TIL signature is associated with survival benefit in patients treated with immune checkpoint blockers characterized by elevated TILs with low activation and proliferation. We further demonstrate that dormant TILs can be reinvigorated upon PD-1 blockade in a patient-derived xenograft model.

Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner.

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells “home” to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1–141) and PTH(1–34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.