Proteinoid Polymers and Nanocapsules for Cancer Diagnostics, Therapy and Theranostics: In Vitro and In Vivo Studies.

Proteinoids-simple polymers composed of amino acids-were suggested decades ago by Fox and coworkers to form spontaneously by heat. These special polymers may self-assemble in micrometer structures called proteinoid microspheres, presented as the protocells of life on earth. Interest in proteinoids increased in recent years, in particular for nano-biomedicine. They were produced by stepwise polymerization of 3-4 amino acids. Proteinoids based on the RGD motif were prepared for targeting tumors. Nanocapsules form by heating proteinoids in an aqueous solution and slowly cooling to room temperature. Proteinoid polymers and nanocapsules suit many biomedical applications owing to their non-toxicity, biocompatibility and immune safety. Drugs and/or imaging reagents for cancer diagnostic, therapeutic and theranostic applications were encapsulated by dissolving them in aqueous proteinoid solutions. Here, recent in vitro and in vivo studies are reviewed.

Three-month follow-up of durability of response to the third dose of the SARS-CoV-2 BNT162b2 vaccine in adults aged 60 years and older: a prospective cohort study.

OBJECTIVE: To evaluate the durability of response 3 months after the third BNT162b2 vaccine in adults aged 60 years and older. DESIGN: Prospective cohort study. SETTING: Single tertiary centre. PARTICIPANTS: Healthcare workers/family members aged ≥60 years old who received the third BNT162b2 dose. INTERVENTIONS: Blood samples were drawn immediately before (T0), 10-19 days (T1) and 74-103 days (T2) after the third dose. PRIMARY AND SECONDARY OUTCOME MEASURES: Anti-spike IgG titres were determined using a commercial assay and seropositivity was defined as ≥50 arbitrary units (AU)/mL. Neutralising antibody titres were determined at T2. Adverse events, COVID-19 infections and Clinical Frailty Scale (CFS) levels were documented. RESULTS: The analysis included 97 participants (median age, 70 years (IQR, 66-74), 58% CFS level 2). IgG titres, which increased significantly from T0 to T1 (median, 440 AU/mL (IQR, 294-923) and median, 25 429 AU/mL (IQR, 14 203-36 114), respectively; p<0.001), decreased significantly by T2, but all remained seropositive (median, 8306 AU/mL (IQR, 4595-14 701), p<0.001 vs T1). In a multivariable analysis, only time from the second vaccine was significantly associated with lower IgG levels at T2 (p=0.017). At T2, 60 patients were evaluated for neutralising antibodies; all were seropositive (median, 1294 antibody titres; IQR, 848-2072). Neutralising antibody and anti-spike IgG levels were correlated (r=0.6, p<0.001). No major adverse events or COVID-19 infections were reported. CONCLUSIONS: Anti-spike IgG and neutralising antibody levels remain adequate 3 months after the third BNT162b2 vaccine in healthy adults aged ≥60 years, although the decline in IgG is concerning. A third dose of vaccine in this population should be top priority.

Humoral and T-Cell Response before and after a Fourth BNT162b2 Vaccine Dose in Adults ≥60 Years.

Both humoral and cellular anamnestic responses are significant for protective immunity against SARS-CoV-2. In the current study, the responses in elderly people before and after a fourth vaccine dose of BNT162b2 were compared to those of individuals immunized with three vaccine doses. Although a boost effect was observed, the high response following the third administration questions the necessity of an early fourth boost.

Palbociclib in combination with sunitinib exerts a synergistic anti-cancer effect in patient-derived xenograft models of various human cancers types.

The efficacy/safety of combining palbociclib (a CDK4/6 inhibitor) and sunitinib (a multi-targeted receptor tyrosine kinase inhibitor) was evaluated, using patient-derived xenograft (PDX) models. Twenty-three PDX mice models were developed from patients with various solid tumors. The mice were randomized to 4 groups (5-6 mice in each): control/palbociclib (100 mg/kg)/sunitinib (50 mg/kg)/combination. Drugs were administered orally, 5 days/week. In 17/23 PDX models (74%), the combination demonstrated a synergistic inhibitory effect vs the monotherapies ("responder" models) with no unexpected toxicities. In 13/17 responder models, where standard-of-care (SOC) was an additional comparator, the combination was more effective than SOC in 7 models, as effective in 4, and less effective in 2. The mean ± SEM experiment duration in 15/17 responder models (2/17 were excluded due to technical issues) was 86 ± 12 and 31 ± 5 days for the combination and control groups, respectively (p = 0.0002). The effect of the combination was dose-dependent. Cell-viability experiments in A549/MDA-MB-231/HT-29 cell lines and experiments using tumor-derived primary cell spheroids supported the PDX findings. In conclusion, combination of palbociclib and sunitinib exerts a synergistic anti-tumor effect without adding unexpected toxicity. A clinical trial assessing this combination is underway.

A genome-wide CRISPR activation screen reveals Hexokinase 1 as a critical factor in promoting resistance to multi-kinase inhibitors in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

Tumor-Targeted Fluorescent Proteinoid Nanocapsules Encapsulating Synergistic Drugs for Personalized Cancer Therapy.

Personalized cancer treatment based on specific mutations offers targeted therapy and is preferred over "standard" chemotherapy. Proteinoid polymers produced by thermal step-growth polymerization of amino acids may form nanocapsules (NCs) that encapsulate drugs overcoming miscibility problems and allowing passive targeted delivery with reduced side effects. The arginine-glycine-glutamic acid (RGD) sequence is known for its preferential attraction to αvß3 integrin, which is highly expressed on neovascular endothelial cells that support tumor growth. Here, tumor-targeted RGD-based proteinoid NCs entrapping a synergistic combination of Palbociclib (Pal) and Alpelisib (Alp) were synthesized by self-assembly to induce the reduction of tumor cell growth in different types of cancers. The diameters of the hollow and drug encapsulating poly(RGD) NCs were 34 ± 5 and 22 ± 3 nm, respectively; thereby, their drug targeted efficiency is due to both passive and active targeting. The encapsulation yield of Pal and Alp was 70 and 90%, respectively. In vitro experiments with A549, MCF7 and HCT116 human cancer cells demonstrate a synergistic effect of Pal and Alp, controlled release and dose dependence. Preliminary results in a 3D tumor spheroid model with cells derived from patient-derived xenografts of colon cancer illustrate disassembly of spheroids, indicating that the NCs have therapeutic potential.

Evaluation of Seropositivity Following BNT162b2 Messenger RNA Vaccination for SARS-CoV-2 in Patients Undergoing Treatment for Cancer.

Importance: Patients with cancer undergoing treatment are at high risk of COVID-19 following SARS-CoV-2 infection; however, their ability to produce an adequate antibody response to messenger RNA SARS-CoV-2 vaccines is unclear. Objective: To evaluate rates of antispike (anti-S) antibody response to a BNT162b2 vaccine in patients with cancer who are undergoing systemic treatment vs healthy controls. Design, Setting, and Participants: This prospective cohort study included 102 adult patients with solid tumors undergoing active intravenous anticancer treatment and 78 controls who received the second dose of the BNT162b2 vaccine at least 12 days before enrollment. The controls were taken from a convenience sample of the patients' family/caregivers who accompanied them to treatment. The study was conducted between February 22, 2021, and March 15, 2021 at Davidoff Cancer Center at Beilinson Hospital (Petah Tikva, Israel). Interventions: Blood samples were drawn from the study participants. Serum samples were analyzed and the titers of the IgG antibodies against SARS-CoV-2 spike receptor-binding domain were determined using a commercially available immunoassay. Seropositivity was defined as 50 or greater AU/mL. Main Outcomes and Measures: The primary outcome was the rate of seropositivity. Secondary outcomes included comparisons of IgG titers and identifying factors that were associated with seropositivity using univariate/multivariable analyses. Results: The analysis included 180 participants, which comprised 102 patients with cancer (median [interquartile range (IQR)] age, 66 [56-72] years; 58 men [57%]) and 78 healthy controls (median [IQR] age, 62 [49-70] years; 25 men [32%]). The most common tumor type was gastrointestinal (29 [28%]). In the patient group, 92 (90%) were seropositive for SARS-CoV 2 antispike IgG antibodies after the second vaccine dose, whereas in the control group, all were seropositive. The median IgG titer in the patients with cancer was significantly lower than that in the controls (1931 [IQR, 509-4386] AU/mL vs 7160 [IQR, 3129-11 241] AU/mL; P < .001). In a multivariable analysis, the only variable that was significantly associated with lower IgG titers was treatment with chemotherapy plus immunotherapy (ß, -3.5; 95% CI, -5.6 to -1.5). Conclusions and Relevance: In this cohort study of patients with cancer who were receiving active systemic therapy, 90% of patients exhibited adequate antibody response to the BNT162b2 vaccine, although their antibody titers were significantly lower than those of healthy controls. Further research into the clinical relevance of lower titers and their durability is required. Nonetheless, the data support vaccinating patients with cancer as a high priority, even during therapy.

Correction: Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

[This corrects the article DOI: 10.18632/oncotarget.27438.].

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse.

Personalized cancer immunotherapy targeting patient-specific cancer/testis antigens (CTA) and neoantigens may benefit from large-scale tumor human leukocyte antigen (HLA) peptidome (immunopeptidome) analysis, which aims to accurately identify antigens presented by tumor cells. Although significant efforts have been invested in analyzing the HLA peptidomes of fresh tumors, it is often impossible to obtain sufficient volumes of tumor tissues for comprehensive HLA peptidome characterization. This work attempted to overcome some of these obstacles by using patient-derived xenograft tumors (PDX) in mice as the tissue sources for HLA peptidome analysis. PDX tumors provide a proxy for the expansion of the patient tumor by re-grafting them through several passages to immune-compromised mice. The HLA peptidomes of human biopsies were compared with those derived from PDX tumors. Larger HLA peptidomes were obtained from the significantly larger PDX tumors as compared with the patient biopsies. The HLA peptidomes of different PDX tumors derived from the same source tumor biopsy were very reproducible, even following subsequent passages to new naïve mice. Many CTA-derived HLA peptides were discovered, as well as several potential neoantigens/variant sequences. Taken together, the use of PDX tumors for HLA peptidome analysis serves as a highly expandable and stable source of reproducible and authentic peptidomes, opening up new opportunities for defining large HLA peptidomes when only small tumor biopsies are available. This approach provides a large source for tumor antigens identification, potentially useful for personalized immunotherapy.

Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer.

p53 status in stromal fibroblasts modulates tumor growth in an SDF1-dependent manner.

The p53 tumor suppressor exerts a variety of cell-autonomous effects that are aimed to thwart tumor development. In addition, however, there is growing evidence for cell nonautonomous tumor suppressor effects of p53. In the present study, we investigated the impact of stromal p53 on tumor growth. Specifically, we found that ablation of p53 in fibroblasts enabled them to promote more efficiently the growth of tumors initiated by PC3 prostate cancer-derived cells. This stimulatory effect was dependent on the increased expression of the chemokine SDF-1 in the p53-deficient fibroblasts. Notably, fibroblasts harboring mutant p53 protein were more effective than p53-null fibroblasts in promoting tumor growth. The presence of either p53-null or p53-mutant fibroblasts led also to a markedly elevated rate of metastatic spread of the PC3 tumors. These findings implicate p53 in a cell nonautonomous tumor suppressor role within stromal fibroblasts, through suppressing the production of tumor stimulatory factors by these cells. Moreover, expression of mutant p53 by tumor stroma fibroblasts might exert a gain of function effect, further accelerating tumor development.

Involvement of stromal p53 in tumor-stroma interactions.

p53 is a major tumor-suppressor gene, inactivated by mutations in about half of all human cancer cases, and probably incapacitated by other means in most other cases. Most research regarding the role of p53 in cancer has focused on its ability to elicit apoptosis or growth arrest of cells that are prone to become malignant owing to DNA damage or oncogene activation, i.e. cell-autonomous activities of p53. However, p53 activation within a cell can also exert a variety of effects upon neighboring cells, through secreted factors and paracrine and endocrine mechanisms. Of note, p53 within cancer stromal cells can inhibit tumor growth and malignant progression. Cancer cells that evolve under this inhibitory influence acquire mechanisms to silence stromal p53, either by direct inhibition of p53 within stromal cells, or through pressure for selection of stromal cells with compromised p53 function. Hence, activation of stromal p53 by chemotherapy or radiotherapy might be part of the mechanisms by which these treatments cause cancer regression. However, in certain circumstances, activation of stromal p53 by cytotoxic anti-cancer agents might actually promote treatment resistance, probably through stromal p53-mediated growth arrest of the cancer cells or through protection of the tumor vasculature. Better understanding of the underlying molecular mechanisms is thus required. Hopefully, this will allow their manipulation towards better inhibition of cancer initiation, progression and metastasis.

The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression.

Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.

Transcriptional activation of miR-34a contributes to p53-mediated apoptosis.

p53 is a potent tumor suppressor, whose biological effects are largely due to its function as a transcriptional regulator. Here we report that, in addition to regulating the expression of hundreds of protein-coding genes, p53 also modulates the levels of microRNAs (miRNAs). Specifically, p53 can induce expression of microRNA-34a (miR-34a) in cultured cells as well as in irradiated mice, by binding to a perfect p53 binding site located within the gene that gives rise to miR-34a. Processing of the primary transcript into mature miR-34a involves the excision of a 30 kb intron. Notably, inactivation of miR-34a strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress, whereas overexpression of miR-34a mildly increases apoptosis. Hence, miR-34a is a direct proapoptotic transcriptional target of p53 that can mediate some of p53's biological effects. Perturbation of miR-34a expression, as occurs in some human cancers, may thus contribute to tumorigenesis by attenuating p53-dependent apoptosis.

p53 Attenuates cancer cell migration and invasion through repression of SDF-1/CXCL12 expression in stromal fibroblasts.

The p53 tumor suppressor acts as a major barrier against cancer. To a large extent, this is due to its ability to maintain genome stability and to eliminate cancer cells from the replicative pool through cell-autonomous mechanisms. However, in addition to its well-documented functions within the malignant cancer cell, p53 can also exert non-cell-autonomous effects that contribute to tumor suppression. We now report that p53 can suppress the production of the chemokine SDF-1 in cultured fibroblasts of both human and mouse origin. This is due to a p53-mediated down-regulation of SDF-1 mRNA, which can be exacerbated on activation of p53 by the drug Nutlin-3. SDF-1 promotes the migration and invasiveness of cells that express its cognate receptor CXCR4. Indeed, medium conditioned by p53-deficient fibroblasts induces cancer cells towards increased directional migration and invasiveness, which are largely reversed by CXCR4 antagonist peptides. Because SDF-1 produced by stromal fibroblasts plays an important role in cancer progression and metastasis, our findings suggest that the ability of p53 to suppress stromal SDF-1 production may be an important mechanism whereby it does its non-cell-autonomous tumor suppressor function.