Treatment with decitabine induces the expression of stemness markers, PD-L1 and NY-ESO-1 in colorectal cancer: potential for combined chemoimmunotherapy.

BACKGROUND: The mechanism of tumor immune escape and progression in colorectal cancer (CRC) is widely investigated in-vitro to help understand and identify agents that might play a crucial role in response to treatment and improve the overall survival of CRC patients. Several mechanisms of immune escape and tumor progression, including expression of stemness markers, inactivation of immunoregulatory genes by methylation, and epigenetic silencing, have been reported in CRC, indicating the potential of demethylating agents as anti-cancer drugs. Of these, a chemotherapeutic demethylating agent, Decitabine (DAC), has been reported to induce a dual effect on both DNA demethylation and histone changes leading to an increased expression of target biomarkers, thus making it an attractive anti-tumorigenic drug. METHODS: We compared the effect of DAC in primary 1076 Col and metastatic 1872 Col cell lines isolated and generated from patients' tumor tissues. Both cell lines were treated with DAC, and the expression of the NY-ESO-1 cancer-testis antigen, the PD-L1 immunoinhibitory marker, and the CD44, Nanog, KLF-4, CD133, MSI-1 stemness markers were analyzed using different molecular and immunological assays. RESULTS: DAC treatment significantly upregulated stemness markers in both primary 1076 Col and meta-static 1872 Col cell lines, although a lower effect occurred on the latter: CD44 (7.85 fold; ***p = 0.0001 vs. (4.19 fold; *p = 0.0120), Nanog (4.1 fold; ***p < 0.0001 vs.1.69 fold; ***p = 0.0008), KLF-4 (4.33 fold; ***p < 0.0001 vs.2.48 fold; ***p = 0.0005), CD133 (16.77 fold; ***p = 0.0003 vs.6.36 fold; *p = 0.0166), and MSI-1 (2.33 fold; ***p = 0.0003 vs.2.3 fold; ***p = 0.0004), respectively. Interestingly, in the metastatic 1872 Col cells treated with DAC, the expression of both PD-L1 and NY-ESO-1 was increased tenfold (*p = 0.0128) and fivefold (***p < 0.0001), respectively. CONCLUSIONS: We conclude that the upregulation of both stemness and immune checkpoint markers by DAC treatment on CRC cells might represent a mechanism of immune evasion. In addition, induction of NY-ESO-1 may represent an immuno-therapeutic option in metastatic CRC patients. Finally, the combination of DAC and anti-PD-1/anti-PD-L1 antibodies treatment should represent a potential therapeutic intervention for this group of patients.

Studies on anti-colon cancer potential of nanoformulations of curcumin and succinylated curcumin in mannosylated chitosan.

Colon cancer (CRC) is the second leading cause of death and the third most diagnosed cancer worldwide. Although curcumin (CUR) has demonstrated a potent anticancer activity, it is characterized by its poor solubility, low bioavailability, and instability. This study is a projection from a previous investigation where CUR and succinylated CUR (CUR.SA) were separately encapsulated in mannosylated-chitosan nanoparticles (CM-NPs) to form CUR-NPs and CUR.SA-NPs, respectively. Here, we aim to assess the anti-CRC activity of these two nanoformulations. Cytotoxicity studies using CCK-8 assay indicated that both CUR-NPs and CUR.SA-NPs have a dose and time-dependent toxicity towards CRC human cell-lines (HCT116 and SW480), and more cytotoxic compared to free CUR or CUR-SA in a time-dependent manner. A significant induction of early and late apoptosis in the CUR-NPs and CUR.SA-NPs treated CRC cell lines compared to untreated cells was observed. Western blotting analyses confirmed the induction of apoptosis through activation of Caspase signaling compared to untreated cells. Based on the physicochemical properties of CUR-NPs and CUR.SA-NPs along with the data from the in vitro studies, we may conclude these nanoparticle formulations hold very promising attributes, worthy of further investigations for its role in the management of CRC.

Persistence of spike-specific immune responses in BNT162b2-vaccinated donors and generation of rapid ex-vivo T cells expansion protocol for adoptive immunotherapy: A pilot study.

Introduction: The BNT162b2 mRNA-based vaccine has shown high efficacy in preventing COVID-19 infection but there are limited data on the types and persistence of the humoral and T cell responses to such a vaccine. Methods: Here, we dissect the vaccine-induced humoral and cellular responses in a cohort of six healthy recipients of two doses of this vaccine. Results and discussion: Overall, there was heterogeneity in the spike-specific humoral and cellular responses among vaccinated individuals. Interestingly, we demonstrated that anti-spike antibody levels detected by a novel simple automated assay (Jess) were strongly correlated (r=0.863, P<0.0001) with neutralizing activity; thus, providing a potential surrogate for neutralizing cell-based assays. The spike-specific T cell response was measured with a newly modified T-spot assay in which the high-homology peptide-sequences cross-reactive with other coronaviruses were removed. This response was induced in 4/6 participants after the first dose, and all six participants after the second dose, and remained detectable in 4/6 participants five months post-vaccination. We have also shown for the first time, that BNT162b2 vaccine enhanced T cell responses also against known human common viruses. In addition, we demonstrated the efficacy of a rapid ex-vivo T cell expansion protocol for spike-specific T cell expansion to be potentially used for adoptive-cell therapy in severe COVID-19, immunocompromised individuals, and other high-risk groups. There was a 9 to 13.7-fold increase in the number of expanded T cells with a significant increase of anti-spike specific response showing higher frequencies of both activation and cytotoxic markers. Interestingly, effector memory T cells were dominant in all four participants' CD8+ expanded memory T cells; CD4+ T cells were dominated by effector memory in 2/4 participants and by central memory in the remaining two participants. Moreover, we found that high frequencies of CD4+ terminally differentiated memory T cells were associated with a greater reduction of spike-specific activated CD4+ T cells. Finally, we showed that participants who had a CD4+ central memory T cell dominance expressed a high CD69 activation marker in the CD4+ activated T cells.

Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.

Although many flow cytometers can analyze 30-50 parameters, it is still challenging to develop a 40+ color panel for the phenotyping of immune cells using fluorochrome conjugated antibodies due to limitations in the availability of spectrally unique fluorochromes that can be excited by the commonly used laser lines (UV, Violet, Blue, Green/Yellow-green, and Red). Spectral flowcytometry is capable of differentiating fluorochromes with significant overlap in the emission spectra, enabling the use of spectrally similar fluorochrome pairs such as Brilliant Blue 515 and FITC in a single panel. We have developed a 43 color panel to characterize most of the immune subsets within the peripheral immune system, including conventional T cells, unconventional T cells such as invariant natural killer T cells (iNKT), Gamma delta (γδ) T-cell subsets (TCR Vδ2, TCR Vγ9) and mucosal-associated invariant T cells (MAIT), B-cell subsets, natural killer (NK) cells, plasmacytoid dendritic cells, dendritic cell subsets, hematopoietic progenitor cells, basophils, and innate lymphoid cell (ILC) subsets (CD117, CRTH2). The panel includes surface markers to analyze activation (CD38, HLA-DR, ICOS/CD278), differentiation (CD45RA, CD27, CD28, CD57), expression of cytokine and chemokine receptors (CD25, CD127, CCR10, CCR6, CCR4, CXCR3, CXCR5, CRTH2/CD294), and co-inhibitory molecules and exhaustion (PD-1, CD223/LAG-3, TIGIT), which enables a deep characterization of PBMCs from peripheral blood. Cells were analyzed on a 5-laser Cytek Aurora and data analysis was done using FlowJo. This panel can help to make a thorough interpretation of immune system, specifically when specimen quantity is low. The panel has not been completely optimized but would rather act as a guide toward the development of a workflow for optimized multicolor immunophenotyping panel.

Dysregulated Phosphorylation of p53, Autophagy and Stemness Attributes the Mutant p53 Harboring Colon Cancer Cells Impaired Sensitivity to Oxaliplatin.

Colorectal cancer (CRC) forms one of the highest ranked cancer types in the world with its increasing incidence and mortality rates despite the advancement in cancer therapeutics. About 50% of human CRCs are reported to have defective p53 expression resultant of TP53 gene mutation often contributing to drug resistance. The current study was aimed to investigate the response of wild-type TP53 harboring HCT 116 and mutant TP53 harboring HT 29 colon cancer cells to chemotherapeutic drug oxaliplatin (OX) and to elucidate the underlying molecular mechanisms of sensitivity/resistance in correlation to their p53 status. OX inhibited growth of wild-type p53-harboring colon cancer cells via p53/p21-Bax mediated apoptosis. Our study revealed that dysregulated phosphorylation of p53, autophagy as well as cancer stemness attributes the mutant p53-harboring colon cancer cells impaired sensitivity to OX.

Persistent anti-NY-ESO-1-specific T cells and expression of differential biomarkers in a patient with metastatic gastric cancer benefiting from combined radioimmunotherapy treatment: a case report.

Combined radioimmunotherapy is currently being investigated to treat patients with cancer. Anti-programmed cell death-1 (PD-1) immunotherapy offers the prospect of long-term disease control in solid tumors. Radiotherapy has the ability to promote immunogenic cell death leading to the release of tumor antigens, increasing infiltration and activation of T cells. New York esophageal squamous cell carcinoma-1 (NY-ESO-1) is a cancer-testis antigen expressed in 20% of advanced gastric cancers and known to induce humoral and cellular immune responses in patients with cancer. We report on the dynamic immune response to the NY-ESO-1 antigen and important immune-related biomarkers in a patient with metastatic gastric cancer treated with radiotherapy combined with anti-PD-1 pembrolizumab antibody.Our patient was an 81-year-old man diagnosed with locally advanced unresectable mismatch repair-deficient gastric cancer having progressed to a metastatic state under a second line of systemic treatment consisting of an anti-PD-1 pembrolizumab antibody. The patient was subsequently treated with local radiotherapy administered concomitantly with anti-PD-1, with a complete response on follow-up radiologic assessment. Disease control was sustained with no further therapy for a period of 12 months before relapse. We have identified an NY-ESO-1-specific interferon-γ (IFN-γ) secretion from the patients' T cells that was significantly increased at response (****p˂0.0001). A novel promiscuous immunogenic NY-ESO-1 peptide P39 (P153-167) restricted to the four patient's HLA-DQ and HLA-DP alleles was identified. Interestingly, this peptide contained the known NY-ESO-1-derived HLA-A2-02:01(P157-165) immunogenic epitope. We have also identified a CD107+ cytotoxic T cell subset within a specific CD8+/HLA-A2-NY-ESO-1 T cell population that was low at disease progression, markedly increased at disease resolution and significantly decreased again at disease re-progression. Finally, we identified two groups of cytokines/chemokines. Group 1 contains five cytokines (IFN-γ, tumor necrosis factor-α, interleukin-2 (IL-2), IL-5 and IL-6) that were present at disease progression, significantly downregulated at disease resolution and dramatically upregulated again at disease re-progression. Group 2 contains four biomarkers (perforin, soluble FAS, macrophage inflammatory protein-3α and C-X-C motif chemokine 11/Interferon-inducible T Cell Alpha Chemoattractant that were present at disease progression, significantly upregulated at disease resolution and dramatically downregulated again at disease re-progression. Combined radioimmunotherapy can enhance specific T cell responses to the NY-ESO-1 antigen that correlates with beneficial clinical outcome of the patient.

Sanguinarine Induces Apoptosis Pathway in Multiple Myeloma Cell Lines via Inhibition of the JaK2/STAT3 Signaling.

Sanguinarine (SNG), a benzophenanthridine alkaloid, has displayed various anticancer abilities in several vivo and in vitro studies. However, the anticancer potential of SNG is yet to be established in multiple myeloma (MM), a mostly incurable malignancy of plasma cells. In this study, we aimed to investigate the potential anti-proliferative and pro-apoptotic activities of SNG in a panel of MM cell lines (U266, IM9, MM1S, and RPMI-8226). SNG treatment of MM cells resulted in a dose-dependent decrease in cell viability through mitochondrial membrane potential loss and activation of caspase 3, 9, and cleavage of PARP. Pre-treatment of MM cells with a universal caspase inhibitor, Z-VAD-FMK, prevented SNG mediated loss of cell viability, apoptosis, and caspase activation, confirming that SNG-mediated apoptosis is caspase-dependent. The SNG-mediated apoptosis appears to be resulted from suppression of the constitutively active STAT3 with a concomitant increase in expression of protein tyrosine phosphatase (SHP-1). SNG treatment of MM cells leads to down-regulation of the anti-apoptotic proteins including cyclin D, Bcl-2, Bclxl, and XIAP. In addition, it also upregulates pro-apoptotic protein, Bax. SNG mediated cellular DNA damage in MM cell lines by induction of oxidative stress through the generation of reactive oxygen species and depletion of glutathione. Finally, the subtoxic concentration of SNG enhanced the cytotoxic effects of anticancer drugs bortezomib (BTZ) by suppressing the viability of MM cells via induction of caspase-mediated apoptosis. Altogether our findings demonstrate that SNG induces mitochondrial and caspase-dependent apoptosis, generates oxidative stress, and suppresses MM cell lines proliferation. In addition, co-treatment of MM cell lines with sub-toxic doses of SNG and BTZ potentiated the cytotoxic activity. These results would suggest that SNG could be developed into therapeutic agent either alone or in combination with other anticancer drugs in MM.