Doxorubicin loaded thermostable nanoarchaeosomes: a next-generation drug carrier for breast cancer therapeutics.

Breast cancer has a poor prognosis due to the toxic side effects associated with high doses of chemotherapy. Liposomal drug encapsulation has resulted in clinical success in enhancing chemotherapy tolerability. However, the formulation faces severe limitations with a lack of colloidal stability, reduced drug efficiency, and difficulties in storage conditions. Nanoarchaeosomes (NA) are a new generation of highly stable nanovesicles composed of the natural ether lipids extracted from archaea. In our study, we synthesized and characterized the NA, evaluated their colloidal stability, drug release potential, and anticancer efficacy. Transmission electron microscopy images have shown that the NA prepared from the hyperthermophilic archaeon Aeropyrum pernix K1 was in the size range of 61 ± 3 nm. The dynamic light scattering result has confirmed that the NA were stable at acidic pH (pH 4) and high temperature (70 °C). The NA exhibited excellent colloidal stability for 50 days with storage conditions at room temperature. The cell viability results have shown that the pure NA did not induce cytotoxicity in NIH 3T3 fibroblast cells and are biocompatible. Then NA were loaded with doxorubicin (NAD), and FTIR and UV-vis spectroscopy results have confirmed high drug loading efficiency of 97 ± 1% with sustained drug release for 48 h. The in vitro cytotoxicity studies in MCF-7 breast cancer cell lines showed that NAD induced cytotoxicity at less than 10 nM concentration. Fluorescence-activated cell sorting (FACS) results confirmed that NAD induced late apoptosis in nearly 92% of MCF-7 cells and necrosis in the remaining cells with cell cycle arrest at the G0/G1 phase. Our results confirmed that the NA could be a potential next-generation carrier with excellent stability, high drug loading efficiency, sustained drug release ability, and increased therapeutic efficacy, thus reducing the side effects of conventional drugs.

Elucidating shape-mediated drug carrier mechanics of hematite nanomaterials for breast cancer therapeutics.

Metallic nanomaterials have gained significant attention in cancer therapy as potential nanocarriers due to their unique properties at the nanoscale. However, nanomaterials face several drawbacks, including biocompatibility, stability, and cellular uptake. Hematite (α-Fe2O3) nanoparticles are emerging as promising nano-carriers to reduce adverse outcomes of conventional chemotherapeutics. However, the shape-mediated drug carrier mechanics of hematite nanomaterials are not raveled. In this study, we tailored hematite nanoparticles in ellipsoidal (EHNP) and spherical (SHNP) shapes with excellent biocompatibility and efficient drug encapsulation and release. We elucidate that EHNP exhibits higher cellular uptake than SHNP. With effective cellular internalization, the cisplatin-loaded EHNP showed excellent cytotoxicity with an IC50 value of 200 nM compared to the cisplatin-loaded SHNP. The flow cytometry cell sorting (FACS) analysis showed a four-fold increase in cell death by arresting the cells at the G0/G1 and G1 phases for cis-EHNP compared to cis-SHNP. The results show that ellipsoidal-shaped hematite nanoparticles can act as attractive nanocarriers with improved therapeutic efficacy in cancer therapy.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Combination of IDO1high and CCL19low expression in the tumor tissue reduces survival in HPV positive cervical cancer.

The over expression of Indoleamine 2, 3-Dioxygenase (IDO1), an immune checkpoint inhibitor, is well known in cervical cancer. However, its association with chemokine signals promoting cellular infiltration in the cervical tumor microenvironment, is unknown. In the current study, we evaluated the expression and enzymatic activity of IDO1. We also profiled the expression of chemokine ligand-receptors- CCR4-CCL22, CXCR3-CXCL10, CXCR4-CXCL12, and CCR7-CCL19 using immunohistochemistry (IHC), and studied their association with IDO1, statistically. After getting an informed consent, punch biopsy samples were obtained from 105 patients diagnosed with cervical cancer. HPV typing by Sanger sequencing, realtime PCR for quantifying IDO1 mRNA expression, HPLC for determining the K/T ratio and IHC for all the above chemokine receptor-ligand pairs along with IDO1 were performed. We found a significant increase in the expression of IDO1 and K/T levels in early and locally advanced stages when compared to Stage IV disease. Among the chemokine ligand -receptor pairs profiled, we found that high CCL19 marker expression was a good prognostic indicator of patients' disease-free (p = 0.013) and overall survival (p = 0.043). Although we could not identify IDO1 as an independent prognostic factor, we found that high levels of IDO1 expression may further reduce survival outcomes in patients with low CCL19 expression. This could be vital for designing immuno therapeutic interventions targeting IDO1.

Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. METHODS: Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. RESULTS: Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. CONCLUSIONS: In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention.

Autologous cervical tumor lysate pulsed dendritic cell stimulation followed by cisplatin treatment abrogates FOXP3+ cells in vitro.

OBJECTIVE: Dendritic cells (DCs) are administered as immunotherapeutic adjuvants after the completion of standard treatment in most settings. However, our Phase I trial indicated that one patient out of four, who received autologous tumor lysate-pulsed dendritic cell (TLDC) also received cisplatin chemotherapy and experienced complete regression of her lung lesion, continuing to be disease free till date. Hence, the objective of our current study is to evaluate the sustenance or augmentation of immune responses when autologous human papillomavirus positive cervical tumor lysate pulsed DC- are combined with cisplatin, using co-culture assays in vitro. METHODS: Before treatment, peripheral blood and punch biopsy samples were collected from 23 cervical cancer patients after obtaining an informed consent. DC functionality was confirmed through phenotypic and functional assays using autologous peripheral blood mononuclear cells as responders. For cisplatin experiments, the drug was added at 150, 200 (clinical dose equivalent), and 400 µM concentrations to DCs alone or DC-T cell co-cultures. Phenotypic assessment and functional characterization of DCs was done using flow cytometry. Cytokine enzyme-linked immunosorbent assay and interferon (IFN)-γ enzyme-linked immune absorbent spot assays were also performed. RESULTS: The functionality of TLDCs was not compromised upon cisplatin treatment in vitro even at the highest (400 µM) concentration. Even though cisplatin treatment reduced the secretion of IFN-γ and interleukin (IL)-12p40 in co-cultures stimulated with TLDCs, this effect was not significant (p>0.05). A doubling of IFN-γ secretion following cisplatin treatment was observed in at least one of three independent experiments. Additional experiments showed a reduction in both FOXP3+ regulatory T cells and IL-10 levels. CONCLUSION: Our results provide evidence that cisplatin treatment may be given after autologous TLDC administration to maintain or improve a productive anti-tumor response in vaccinated patients.

Immunotherapy for cervical cancer: Can it do another lung cancer?

Cervical cancer, although preventable, is still the second most common cancer among women worldwide. In developing countries like India, where screening for cervical cancer is virtually absent, most women seek treatment only at advanced stages of the disease. Although standard treatment is curative in more than 90% of women during the early stages, for stage IIIb and above this rate drops to 50% or less. Hence, novel therapeutic adjuvants are required to improve survival at advanced stages. Lung cancer has shown the way forward with the use of Immunotherapeutic interventions as standard line of treatment in advanced stages. In this review, we provide an overview of mechanisms of immune evasion, strategies that can be employed to boost the immune system in order to improve the overall survival of the patients and summarize briefly the clinical trials that have been completed or that are underway to bring therapeutic vaccines for cervical cancer to the clinics.