Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets.

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria.

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

Oncolytic adenoviruses and the treatment of pancreatic cancer: a review of clinical trials.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) remains a common and difficult cancer to treat. Surgical resection and chemotherapy are standard of care and clinical outcomes remain poor. Oncolytic adenoviruses are a unique approach to the treatment of this challenging cancer, aiming to overcome the features of this disease that pose the key obstacles to standard therapies. This paper provides a detailed review of the clinical trials of conditionally-replicative adenoviruses in pancreatic cancer to date, with a brief summary of the past preclinical literature and future prospects of this therapy. METHODS: MEDLINE, Embase, and clinicaltrials.gov were searched from inception to December 23rd 2022 for clinical trials of conditionally-replicative adenoviruses used in patients with pancreatic ductal adenocarcinoma. Primary features for review included patient demographics, treatment protocol including dose and administration route, adverse events, patient responses and survival rates. RESULTS: The six published clinical trials suggest that objective clinical responses can be achieved with a tolerable level of side effects, even at high viral doses. The more clinically adaptable intravenous route of administration also appears to be as well tolerated as the more challenging intratumoural injections. CONCLUSION: Published clinical trials provide data of the safety and some signs of oncolytic activity of conditionally-replicative adenoviruses in patients with pancreatic cancer. Importantly, on the latest trials, the easier intravenous route of administration seems to be well tolerated and safe, providing the opportunity for further clinical evaluation. It is hoped that the ongoing clinical trials will yield more promising results of this therapeutic approach against a currently intractable disease.

CBL0137 impairs homologous recombination repair and sensitizes high-grade serous ovarian carcinoma to PARP inhibitors.

BACKGROUND: High-grade serous ovarian carcinomas (HGSCs) are a heterogeneous subtype of epithelial ovarian cancers and include serous cancers arising in the fallopian tube and peritoneum. These cancers are now subdivided into homologous recombination repair (HR)-deficient and proficient subgroups as this classification impacts on management and prognosis. PARP inhibitors (PARPi) have shown significant clinical efficacy, particularly as maintenance therapy following response to platinum-based chemotherapy in BRCA-mutant or homologous recombination (HR)-deficient HGSCs in both the 1st and 2nd line settings. However, PARPi have limited clinical benefit in HR-proficient HGSCs which make up almost 50% of HGSC and improving outcomes in these patients is now a high priority due to the poor prognosis with ineffectiveness of the current standard of care. There are a number of potential lines of investigation including efforts in sensitizing HR-proficient tumors to PARPi. Herein, we aimed to develop a novel combination therapy by targeting SSRP1 using a small molecule inhibitor CBL0137 with PARPi in HR-proficient HGSCs. EXPERIMENTAL DESIGN: We tested anti-cancer activity of CBL0137 monotherapy using a panel of HGSC cell lines and patient-derived tumor cells in vitro. RNA sequencing was used to map global transcriptomic changes in CBL0137-treated patient-derived HR-proficient HGSC cells. We tested efficacy of CBL0137 in combination with PARPi using HGSC cell lines and patient-derived tumor cells in vitro and in vivo. RESULTS: We show that SSRP1 inhibition using a small molecule, CBL0137, that traps SSRP1 onto chromatin, exerts a significant anti-growth activity in vitro against HGSC cell lines and patient-derived tumor cells, and also reduces tumor burden in vivo. CBL0137 induced DNA repair deficiency via inhibition of the HR repair pathway and sensitized SSRP1-high HR-proficient HGSC cell lines and patient-derived tumor cells/xenografts to the PARPi, Olaparib in vitro and in vivo. CBL0137 also enhanced the efficacy of DNA damaging platinum-based chemotherapy in HGSC patient-derived xenografts. CONCLUSION: Our findings strongly suggest that combination of CBL0137 and PARP inhibition represents a novel therapeutic strategy for HR-proficient HGSCs that express high levels of SSRP1 and should be investigated in the clinic.

Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis.

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3ß/ß-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3ß. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

Immunological Responses to Envenomation.

Venoms are complex mixtures of toxic compounds delivered by bite or sting. In humans, the consequences of envenomation range from self-limiting to lethal. Critical host defence against envenomation comprises innate and adaptive immune strategies targeted towards venom detection, neutralisation, detoxification, and symptom resolution. In some instances, venoms mediate immune dysregulation that contributes to symptom severity. This review details the involvement of immune cell subtypes and mediators, particularly of the dermis, in host resistance and venom-induced immunopathology. We further discuss established venom-associated immunopathology, including allergy and systemic inflammation, and investigate Irukandji syndrome as a potential systemic inflammatory response. Finally, this review characterises venom-derived compounds as a source of immune modulating drugs for treatment of disease.

Venom of the Red-Bellied Black Snake Pseudechis porphyriacus Shows Immunosuppressive Potential.

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

Interaction and molecular dynamics simulation study of Osimertinib (AstraZeneca 9291) anticancer drug with the EGFR kinase domain in native protein and mutated L844V and C797S.

BACKGROUND: Targeted therapy is a novel, promising approach to anticancer treatment that endeavors to overcome drug resistance to traditional chemotherapies. Patients with the L858R mutation in epidermal growth factor receptor (EGFR) respond to the first generation tyrosine kinase inhibitors (TKIs); however, after one year of treatment, they may become resistant. The T790M mutation is the most probable cause for drug resistance. Third generation drugs, including Osimertinib (AZD9291), are more effective against T790M and other sensitive mutations. Osimertinib is effective against the L844V mutation, has conditional effectiveness for the L718Q mutation, and is ineffective for the Cys797Ser (C797S) mutation. Cells that have both the T790M and C797 mutations are more resistant to third generation drugs. Although research has shown that Osimertinib is an effective treatment for EGFR L844V cells, this has not been shown for cells that have the C797S mutation. This molecular mechanism has not been well-studied. METHODS: In the present study, we used the GROMACS software for molecular dynamics simulation to identify interactions between Osimertinib and the kinase part of EGFR in L844V and C797S mutants. RESULTS: We evaluated native EGFR protein and the L844V and C797S mutations' docking and binding energy, kI, intermolecular, internal, and torsional energy parameters. Osimertinib was effective for the EGFR L844V mutation, but not for EGFR C797S. All simulations were validated by root-mean-square deviation (RMSD), root-mean square fluctuation (RMSF), and radius of gyration (ROG). CONCLUSION: According to our computational simulation, the results supported the experimental models and, therefore, could confirm and predict the molecular mechanism of drug efficacy.

CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer.

The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers.

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

Rapid loss of group 1 innate lymphoid cells during blood stage Plasmodium infection.

Objectives: Innate lymphoid cells (ILCs) share many characteristics with CD4+ T cells, and group 1 ILCs share a requirement for T-bet and the ability to produce IFNγ with T helper 1 (Th1) cells. Given this similarity, and the importance of Th1 cells for protection against intracellular protozoan parasites, we aimed to characterise the role of group 1 ILCs during Plasmodium infection. Methods: We quantified group 1 ILCs in peripheral blood collected from subjects infected with with Plasmodium falciparum 3D7 as part of a controlled human malaria infection study, and in the liver and spleens of Pc AS-infected mice. We used genetically-modified mouse models, as well as cell-depletion methods in mice to characterise the role of group 1 ILCs during Pc AS infection. Results: In a controlled human malaria infection study, we found that the frequencies of circulating ILC1s and NK cells decreased as infection progressed but recovered after volunteers were treated with antiparasitic drug. A similar observation was made for liver and splenic ILC1s in P. chabaudi chabaudi AS (Pc AS)-infected mice. The decrease in mouse liver ILC1 frequencies was associated with increased apoptosis. We also identified a population of cells within the liver and spleen that expressed both ILC1 and NK cell markers, indicative of plasticity between these two cell lineages. Studies using genetic and cell-depletion approaches indicated that group 1 ILCs have a limited role in antiparasitic immunity during Pc AS infection in mice. Discussion: Our results are consistent with a previous study indicating a limited role for natural killer (NK) cells during Plasmodium chabaudi infection in mice. Additionally, a recent study reported the redundancy of ILCs in humans with competent B and T cells. Nonetheless, our results do not rule out a role for group 1 ILCs in human malaria in endemic settings given that blood stage infection was initiated intravenously in our experimental models, and thus bypassed the liver stage of infection, which may influence the immune response during the blood stage. Conclusion: Our results show that ILC1s are lost early during mouse and human malaria, and this observation may help to explain the limited role for these cells in controlling blood stage infection.

Immune drug discovery from venoms.

This review catalogues recent advances in knowledge on venoms as standalone therapeutic agents or as blueprints for drug design, with an emphasis on venom-derived compounds that affects the immune system. We discuss venoms and venom-derived compounds that affect total immune cell numbers, immune cell proliferation, immune cell migration, immune cell phenotype and cytokine secretion. Identifying novel compounds that 'tune' the system, up-regulating the immune response during infectious disease and cancer and down-regulating the immune response during autoimmunity, will greatly expand the tool kit of human immunotherapeutics. Targeting these pathways may also open therapeutic options that alleviate symptoms of envenomation. Finally, combining recent advances in venomics with progress in low cost, high-throughput screening platforms will no doubt yield hundreds of prototype immune modulating compounds in the coming years.

Targeted Therapies for Triple-Negative Breast Cancer: Combating a Stubborn Disease.

Triple-negative breast cancers (TNBCs) constitute a heterogeneous subtype of breast cancers that have a poor clinical outcome. Although no approved targeted therapy is available for TNBCs, molecular-profiling efforts have revealed promising molecular targets, with several candidate compounds having now entered clinical trials for TNBC patients. However, initial results remain modest, thereby highlighting challenges potentially involving intra- and intertumoral heterogeneity and acquisition of therapy resistance. We present a comprehensive review on emerging targeted therapies for treating TNBCs, including the promising approach of immunotherapy and the prognostic value of tumor-infiltrating lymphocytes. We discuss the impact of pathway rewiring in the acquisition of drug resistance, and the prospect of employing combination therapy strategies to overcome challenges towards identifying clinically-viable targeted treatment options for TNBC.

Exome Sequencing to Predict Neoantigens in Melanoma.

The ability to use circulating peripheral blood cells and matched tumor sequencing data as a basis for neoantigen prediction has exciting possibilities for application in the personalized treatment of cancer patients. We have used a high-throughput screening approach, combining whole-exome sequence data, mRNA microarrays, and publicly available epitope prediction algorithm output to identify mutated proteins processed and displayed by patient tumors and recognized by circulating immune cells. Matched autologous melanoma cell lines and peripheral blood mononuclear cells were used to create mixed lymphocyte tumor cell cultures, resulting in an expansion of tumor-reactive T cells to use for mutated peptide screening. Five patients were investigated, three of whom had a durable complete response (CR; 15+ years) in an autologous melanoma-pulsed dendritic cell clinical trial. We identified seven mutated antigens in total that stimulated T-effector memory cells in two of the five patients. While the procedure did not result in clinically applicable neoantigens for all patients, those identified were likely important in tumor clearance, leading to durable CR. The nature of the screening process allows results to be obtained rapidly and is easily applicable to a wide variety of different tumor types.

Apoptosis and dysfunction of blood dendritic cells in patients with falciparum and vivax malaria.

Malaria causes significant morbidity worldwide and a vaccine is urgently required. Plasmodium infection causes considerable immune dysregulation, and elicitation of vaccine immunity remains challenging. Given the central role of dendritic cells (DCs) in initiating immunity, understanding their biology during malaria will improve vaccination outcomes. Circulating DCs are particularly important, as they shape immune responses in vivo and reflect the functional status of other subpopulations. We performed cross-sectional and longitudinal assessments of the frequency, phenotype, and function of circulating DC in 67 Papuan adults during acute uncomplicated P. falciparum, P. vivax, and convalescent P. falciparum infections. We demonstrate that malaria patients display a significant reduction in circulating DC numbers and the concurrent accumulation of immature cells. Such alteration is associated with marked levels of spontaneous apoptosis and impairment in the ability of DC to mature, capture, and present antigens to T cells. Interestingly, sustained levels of plasma IL-10 were observed in patients with acute infection and were implicated in the induction of DC apoptosis. DC apoptosis was reversed upon IL-10 blockade, and DC function recovered when IL-10 levels returned to baseline by convalescence. Our data provide key information on the mechanisms behind DC suppression during malaria and will assist in developing strategies to better harness DC's immunotherapeutic potential.

In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.

Low-level Plasmodium falciparum blood-stage infection causes dendritic cell apoptosis and dysfunction in healthy volunteers.

BACKGROUND: Dendritic cells (DCs) are highly specialized antigen-presenting cells that are crucial for initiation of immune responses. During naturally acquired malaria, DC number and function is reduced. METHODS: The timing of, parasitemia threshold of, and contribution of apoptosis to DC loss were prospectively evaluated in 10 men after experimental challenge with approximately 1800 Plasmodium falciparum-parasitized red blood cells (pRBCs) and after drug cure initiated at a parasite level of ≥ 1000 parasites/mL. RESULTS: The nadir levels of total, myeloid, and plasmacytoid DCs occurred 8 days after infection. DC loss was partially attributable to apoptosis, which was first detected on day 5 (median parasite level, 238 parasites/mL) and maximal at day 7. Remaining DCs exhibited a reduced ability to uptake particulate antigen. DC numbers recovered approximately 60 hours after antimalarial drug administration. There was no loss of DC number or function before or after drug cure in 5 men inoculated with <180 pRBCs and treated on day 6, when their parasite level was approximately 200 parasites/mL. CONCLUSIONS: Plasmodium causes DC loss in vivo, which is at least partially explained by apoptosis in response to blood-stage parasites. In primary infection, loss of DC number and function occurs early during the prepatent period and before or with onset of clinical symptoms. These findings may explain in part the inadequate development of immunity to blood-stage malaria infection.

Proteomic comparison of mcf-7 tumoursphere and monolayer cultures.

Breast cancer is a heterogeneous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in "tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.

Prevalence and prognostic significance of secondary lymphedema following breast cancer.

BACKGROUND: The adverse consequences of lymphedema following breast cancer in relation to physical function and quality of life are clear; however, its potential relationship with survival has not been investigated. Our purpose was to determine the prevalence of lymphedema and associated upper-body symptoms at 6 years following breast cancer and to examine the prognostic significance of lymphedema with respect to overall 6-year survival (OS). METHODS AND RESULTS: A population-based sample of Australian women (n = 287) diagnosed with invasive, unilateral breast cancer was followed for a median of 6.6 years and prospectively assessed for lymphedema (using bioimpedance spectroscopy [BIS], sum of arm circumferences [SOAC], and self-reported arm swelling), a range of upper-body symptoms, and vital status. OS was measured from date of diagnosis to date of death or last follow-up. Kaplan-Meier methods were used to calculate OS and Cox proportional hazards models quantified the risk associated with lymphedema. Approximately 45% of women had reported at least one moderate to extreme symptom at 6.6 years postdiagnosis, while 34% had shown clinical evidence of lymphedema, and 48% reported arm swelling at least once since baseline assessment. A total of 27 (9.4%) women died during the follow-up period, and lymphedema, diagnosed by BIS or SOAC between 6-18 months postdiagnosis, predicted mortality (BIS: HR = 2.5; 95% CI: 0.9, 6.8, p = 0.08; SOAC: 3.0; 95% CI: 1.1, 8.7, p = 0.04). There was no association (HR = 1.2; 95% CI: 0.5, 2.6, p = 0.68) between self-reported arm swelling and OS. CONCLUSIONS: These findings suggest that lymphedema may influence survival following breast cancer treatment and warrant further investigation in other cancer cohorts and explication of a potential underlying biology.

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments.

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.