Introduction of an Ultraviolet C-Irradiated 4T1 Murine Breast Cancer Whole-Cell Vaccine Model.

The advent of immunotherapy has revolutionized cancer treatments. However, the application of immune checkpoint inhibitors may entail severe side effects, with the risk of therapeutic resistance. The generation of chimeric antigen receptor (CAR) T-cells or CAR-NK cells requires specialized molecular laboratories, is costly, and is difficult to adapt to the rapidly growing number of cancer patients. To provide a simpler but effective immune therapy, a whole-cell tumor vaccine protocol was established based on ultraviolet C (UCV)-irradiated 4T1 triple-negative breast cancer cells. The apoptosis of tumor cells after UVC irradiation was verified using resazurin and Annexin V/propidium iodide flow cytometric assays. Protective immunity was achieved in immunized BALB/c mice, showing partial remission. Adoptive transfer of splenocytes or plasma from the mice in remission showed a protective effect in the naive BALB/c mice that received a living 4T1 tumor cell injection. 4T1-specific IgG antibodies were recorded in the plasma of the mice following immunization with the whole-cell vaccine. Interleukin-2 (IL-2) and oligonucleotide 2006 (ODN2006) adjuvants were used for the transfer of splenocytes from C57BL/6 mice into cyclophosphamide-treated BALB/c mice, resulting in prolonged survival, reduced tumor growth, and remission in 33% of the cases, without the development of the graft-versus-host disease. Our approach offers a simple, cost-effective whole-cell vaccine protocol that can be administered to immunocompetent healthy organisms. The plasma or the adoptive transfer of HLA-matching immunized donor-derived leukocytes could be used as an immune cell therapy for cancer patients.

Patient-specific Boolean models of signalling networks guide personalised treatments.

Prostate cancer is the second most occurring cancer in men worldwide. To better understand the mechanisms of tumorigenesis and possible treatment responses, we developed a mathematical model of prostate cancer which considers the major signalling pathways known to be deregulated. We personalised this Boolean model to molecular data to reflect the heterogeneity and specific response to perturbations of cancer patients. A total of 488 prostate samples were used to build patient-specific models and compared to available clinical data. Additionally, eight prostate cell line-specific models were built to validate our approach with dose-response data of several drugs. The effects of single and combined drugs were tested in these models under different growth conditions. We identified 15 actionable points of interventions in one cell line-specific model whose inactivation hinders tumorigenesis. To validate these results, we tested nine small molecule inhibitors of five of those putative targets and found a dose-dependent effect on four of them, notably those targeting HSP90 and PI3K. These results highlight the predictive power of our personalised Boolean models and illustrate how they can be used for precision oncology.

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines.

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Candida albicans Enhances the Progression of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells.

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model.

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer.

Enantioselective Synthesis of 8-Hydroxyquinoline Derivative, Q134 as a Hypoxic Adaptation Inducing Agent.

Hypoxia is a common feature of neurodegenerative diseases, including Alzheimer's disease that may be responsible for disease pathogenesis and progression. Therefore, the hypoxia-inducible factor (HIF)1 system, responsible for hypoxic adaptation, is a potential therapeutic target to combat these diseases by activators of cytoprotective protein induction. We have selected a candidate molecule from our cytoprotective hydroxyquinoline library and developed a novel enantioselective synthesis for the production of its enantiomers. The use of quinidine or quinine as a catalyst enabled the preparation of enantiomer-pure products. We have utilized in vitro assays to evaluate cytoprotective activity, a fluorescence-activated cell sorting (FACS) based assay measuring mitochondrial membrane potential changes, and gene and protein expression analysis. Our data showed that the enantiomers of Q134 showed potent and similar activity in all tested assays. We have concluded that the enantiomers exert their cytoprotective activity via the HIF1 system through HIF1A protein stabilization.

Signatures of cell death and proliferation in perturbation transcriptomics data-from confounding factor to effective prediction.

Transcriptional perturbation signatures are valuable data sources for functional genomics. Linking perturbation signatures to screenings opens the possibility to model cellular phenotypes from expression data and to identify efficacious drugs. We linked perturbation transcriptomics data from the LINCS-L1000 project with cell viability information upon genetic (Achilles project) and chemical (CTRP screen) perturbations yielding more than 90 000 signature-viability pairs. An integrated analysis showed that the cell viability signature is a major factor underlying perturbation signatures. The signature is linked to transcription factors regulating cell death, proliferation and division time. We used the cell viability-signature relationship to predict viability from transcriptomics signatures, and identified and validated compounds that induce cell death in tumor cell lines. We showed that cellular toxicity can lead to unexpected similarity of signatures, confounding mechanism of action discovery. Consensus compound signatures predicted cell-specific drug sensitivity, even if the signature is not measured in the same cell line, and outperformed conventional drug-specific features. Our results can help in understanding mechanisms behind cell death and removing confounding factors of transcriptomic perturbation screens. To interactively browse our results and predict cell viability in new gene expression samples, we developed CEVIChE (CEll VIability Calculator from gene Expression; https://saezlab.shinyapps.io/ceviche/).

Single Cell Mass Cytometry of Non-Small Cell Lung Cancer Cells Reveals Complexity of In vivo And Three-Dimensional Models over the Petri-dish.

Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.

Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations.

Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5⁻10.8 µM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.

Synthesis, cytotoxic characterization, and SAR study of imidazo[1,2-b]pyrazole-7-carboxamides.

The synthesis and in vitro cytotoxic characteristics of new imidazo[1,2-b]pyrazole-7-carboxamides were investigated. Following a hit-to-lead optimization exploiting 2D and 3D cultures of MCF-7 human breast, 4T1 mammary gland, and HL-60 human promyelocytic leukemia cancer cell lines, a 67-membered library was constructed and the structure-activity relationship (SAR) was determined. Seven synthesized analogues exhibited sub-micromolar activities, from which compound 63 exerted the most significant potency with a remarkable HL-60 sensitivity (IC50 = 0.183 µM).

Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

Mannich Curcuminoids as Potent Anticancer Agents.

A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen-Schmidt condensation sequence. Structure-activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues. Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells.

There is an unmet medical need for the improvement of pancreatic islet maintenance in culture. Due to restricted donor availability it is essential to ameliorate islet viability and graft engraftment. The aim of this study was to compare the standard tissue culture techniques with the advanced Real Architecture For 3D Tissue (RAFT™) culture system in terms of viability and hormone production. Here, we first report that islets embedded in RAFT™ collagen type I advanced tissue culture system maintain their tissue integrity better than in monolayer and suspension cultures. The Calcein violet assay and Annexin V/propidium-iodide staining show higher cell viability in the RAFT™ culture system. Quantitative real-time PCR data showed that RAFT™ increases insulin expression after 18 days in culture compared to traditional methods. Enhanced insulin and glucagon production was further verified by immunofluorescent staining in a time-course manner. These results indicate that RAFT™ tissue culture platform can be a promising tool to maintain pancreatic islet spheroid integrity and culture islets for downstream high throughput pharmacological studies ex vivo.

[The potential of three-dimensional tumor models and cell culturing in cancer research and diagnostics]. / A háromdimenziós in vitro tumormodellek jelentõsége a rákkutatásban és diagnosztikában.

In vitro testing of antitumor agents on human cancer cell lines has become essential in pharmaceutical research and in clinical practice. Although the most widely used technique is the two-dimensional cell growing protocol (in tissue culture plates), the new three-dimensional methods are becoming more and more popular as their structure and complexity is more similar to the microenvironment of the real tumor. The aim of the present study is to describe the most widely used in vitro three-dimensional tumor models and to compare a RAFT(TM) three dimensional in vitro tumor model with the traditional two-dimensional tumor cell cultures. In the study, the viability and the enzyme activity of cultured A549 non-small cell lung cancer (NSCLC) cells under different conditions were compared. The results show that while the number of necrotic cells increased significantly (20-fold; 2D/A549 T75 conventional tissue culture flask 1.6%; 2D/A549-collagen coated T75 tissue culture flask 1.45%, RAFT(TM) 22.11%) during long culturing period in the RAFT(TM) three-dimensional in vitro tumor model, there was no significant difference during the conventional antitumor screening period (3-5 day) compared to the traditional two-dimensional cell cultures. The structure of the tumor cell islets grown with RAFT(TM) is much more complex than that of the traditional two-dimensional cultures. Thus, similarly to the in vivo tumor microenvironment, there is also a collagen matrix in the extracellular space which can have significant effect on the diffusion of the antitumor agents to cells. In conclusion, it can be stated that testing of antitumor agents on tumor cells cultured in three-dimensional systems can be an important complementary method to the traditional two-dimensional in vitro analyses. The results of the new three-dimensional method can be more easily applied in the in vivo analysis and translated into clinical practice.

Overexpression of Hsp27 ameliorates symptoms of Alzheimer's disease in APP/PS1 mice.

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-ß (Aß) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimer's disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.