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The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30

Summary Immune checkpoint therapy to reverse natural killer (NK) and T cell exhaustion has emerged as a promising treatment in various cancers. While anti-programmed cell death 1 (PD-1) pembrolizumab has recently gained Food and Drug Administration (FDA) approval for use in recurrent or metastatic cervical cancer, other checkpoint molecules, such as T cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) and T cell immunoglobulin and mucin-domain containing-3 (Tim-3), have yet to be fully explored in this disease. We report expression of TIGIT, Tim-3 and PD-1 on subsets of peripheral blood NK (CD56dim/negCD16bright/dim/neg and CD56brightCD16dim/neg) and T cells. The percentages of these cells were increased in women with cervical cancer and pre-malignant lesions. PD-1+ NK and T cells were likely to co-express TIGIT and/or Tim-3. These cells, with an apparently ‘exhausted’ phenotype, were augmented in patients. A subset of cells were also natural killer group 2 member D (NKG2D)- and DNAX accessory molecule 1 (DNAM-1)-positive. PD-1int and PD-1high T cells were notably increased in cervical cancer. Soluble programmed cell death ligand 1 (PD-L1) was higher in cancer patient blood versus healthy donors and we observed a positive correlation between sPD-L1 and PD-1+ T cells in women with low-grade lesions. Within the cancer group, there were no significant correlations between sPD-L1 levels and cervical cancer stage. However, when comparing cancer versus healthy donors, we observed an inverse association between sPD-L1 and total T cells and a correlation between sPD-L1 and CD56dim NK cells. Our results may show an overview of the immune response towards pre-cancerous lesions and cervical cancer, perhaps giving an early clue as to whom to administer blocking therapies. The increase of multiple checkpoint markers may aid in identifying patients uniquely responsive to combined antibody therapies.

Herein, we report the development of sandwich type Surface Enhanced Raman Spectroscopy (SERS) immunosensor modified to be zwitterionic for the detection of soluble B7–H6 biomarker in blood serum from cervical cancer patients. Anti-fouling capture SERS substrate of biosensor based on gold (Au) thin film was modified with a self-assembled monolayer of zwitterionic l -cysteine to combat serum fouling and was then conjugated with NKp30 receptor protein to capture the B7–H6 biomarker in blood serum. The SERS nanoprobe based on spiky gold nanoparticles (AuNPs) was functionalized with ATP reporter molecule, that is stable at a wide range of pH, making the SERS signal reliable in complex media. Then, it was conjugated with anti-B7-H6 antibody forming the complex anti-B7-H6@ATP@AuNPs (i.e., SERS nanoprobe). The proposed immunosensor demonstrated high reproducibility for the quantitative detection of soluble tumor biomarker B7–H6 within the range of 10−10 M to 10−14 M with limit of detection (LOD) of 10−14 M or 10.8 fg mL−1, in the cancer patient serum, greatly exceeding (100 fold) the LOD of commercially available ELISA kits. Such low LOD is partially the result of zwitterionic modification which reduces the serum fouling by 55% compared to traditionally used BSA blocked capture substrates (i.e., control). Notably, this immunosensors demonstrated higher accuracy for detecting the B7–H6 biomarker in undiluted blood serum samples from cervical cancer patients and outperforms the currently available analytical techniques, making it reliable for point of care (POC) testing.

Herein, we reported the fabrication of novel peptide-conjugated ligand-targeted nanoliposomes (LTLs) for chemo-photodynamic therapy against HER2-positive breast cancer. The LTL core was utilized for encapsulating doxorubicin (DOX) for chemotherapy, and methylene blue (MB) attached NaYF4:Yb,Er upconversion nanoparticles (UCNPs) for NIR-activated bioimaging and leveraging its visible emission for photoexciting MB for enhanced photodynamic therapy (PDT). The specificity of our LTLs was achieved by conjugating a newly discovered anti-HER2 peptide screened from a phage display peptide library. The high selectivity of the peptide-conjugated LTLs was confirmed by confocal imaging of SKBR-3 (HER2-positive) and MCF-7 (HER2-negative) breast cancer cell lines, illustrating its target-specific nature. The energy transfer from UCNPs to MB was verified, thus enabling the generation of reactive oxygen species upon activation with a 975 nm laser source (0.60 W cm-2) under 5 min continuous excitation. A significant decline in the cell viability by 95% was observed using chemo-photodynamic combinational therapy, whereas for chemo-drug alone and PDT alone, the cell proliferation declined by 77% and 84%, respectively. Furthermore, we demonstrated an improved uptake of the LTLs inside a 3D model of SKBR-3 tumor spheroids, where the spheroid cell viability was suppressed by 66% after the use of combinational therapy. Thus, our results suggest great prospective use of theranostic LTLs for breast cancer management.

Nanobodies (Nb) have a promising future as a part of next generation chemodrug delivery systems. Nb, or VHH, are small (15 kDa) monomeric antibody fragments consisting of the antigen binding region of heavy chain antibodies. Heavy chain antibodies are naturally produced by camelids, however the structure of their VHH regions can be readily reproduced in industrial expression systems, such as bacteria or yeast. Due to their small size, high solubility, remarkable stability, manipulatable characteristics, excellent in vivo tissue penetration, conjugation advantages, and ease of production, Nb have many advantages when compared against their antibody precursors. In this review, we discuss the generation and selection of Nbs via phage display libraries for easy screening, and the conjugation techniques involved in creating target-specific nanocarriers. Furthermore, we provide a comprehensive overview of recent developments and perspectives in the field of Nb drug conjugates (NDCs) and Nb-based drug vehicles (NDv) with respect to antitumor therapeutics.

BackgroundAlthough great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored.MethodsIn this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy.ResultsNotably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression.ConclusionsOur results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.

Methylene Blue (MB) based Photodynamic therapy (PDT) has gained much interest recently due to its FDA approval for treating various diseased conditions in human. Herein, we report for the first time the systematic evaluation of NIR-controlled PDT efficiency of free MB and MB attached on NaYF4:Yb, Er nanoparticles (UCNPs) i.e., MB@UCNPs encapsulated within Liposomes (LPs). The UCNPs act as an upconverting energy source for the photosensitizers dye MB for enhanced ROS generation and utilizing the remnant energy for selective tracking or bioimaging. The specificity of our nano LPs system was achieved by conjugating our newly discovered anti-HER2 peptides by phage display technique and demonstrated its specificity by confocal imaging of HER-2 overexpressing in vitro model. A significant decline in the cell viability by 83% and 64% was achieved by the LPs containing MB@UCNPs and free MB with UCNPs, respectively after 975 nm NIR laser exposure (0.60 W/cm−2) for 5 min continuous exposure. Also, the flowcytometry assay confirmed that enhanced ROS generation capability of LPs with MB@UCNPs was higher than the free MB with UCNPs, which states the relevance of the minimal distance between MB and UCNPs for higher anti-cancer efficiency.

Herein, we reported the fabrication of novel peptide-conjugated ligand-targeted nanoliposomes (LTLs) for chemo-photodynamic therapy against HER2-positive breast cancer. The LTL core was utilized for encapsulating doxorubicin (DOX) for chemotherapy, and methylene blue (MB) attached NaYF

Herein, we report the simple and inexpensive approach for the large-scale fabrication of uniform bottom-up Surface Enhanced Raman Spectroscopy (SERS) substrate. SERS substrate was fabricated by controlled sputtering of 10 nm thick gold film on self-assembled silica nanoparticles (SiNPs) of ~120 nm on glass substrates. The SERS detection has been firstly demonstrated using Rhodamine B as a Raman probe molecule with a detection limit of 10−10 M on Au sputtered SiNPs (i.e., Au@SiNPs). The experimental Raman enhancement from 0 to 6 was achieved on Au@SiNPs due to the generation of multiple SERS hotspot. To combat blood serum fouling, the zwitterionic modification of l -cysteine was done on Au@SiNPs substrates which lowered blood serum fouling by 48%. Our SERS-based sensor demonstrated high reproducibility for the detection of Doxorubicin in undiluted blood serum with a limit of detection of 20 nM, which greatly exceeded the detection range of available methodologies. We envision that the translation of this SERS substrate for the detection of chemo-drugs like Doxorubicin will assist clinicians in making rapid and/or early decisions in patients undergoing sustained chemotherapy to lower its side-effects or to incorporate other treatment methodologies as an option for Personalized treatment.