Evaluation of tumor response to immune checkpoint inhibitors by a 3D immunotumoroid model.

Background: Only 20 percent of renal and bladder cancer patients will show a significant response to immune checkpoint inhibitor (ICI) therapy, and no test currently available accurately predicts ICI response. Methods: We developed an "immunotumoroid" cell model system that recapitulates the tumor, its microenvironment, and necessary immune system components in patient-derived spheroids to enable ex vivo assessment of tumor response to ICI therapy. Immunotumoroids were developed from surgically resected renal cell carcinomas and bladder carcinomas selected for high tumor-infiltrating lymphocytes (TILs) and survived more than a month without media exchange. Immunohistochemistry was used to detect immune and non-immune cells in cryopreserved source tumors and the resulting immunotumoroids. Immunotumoroid response to ICIs (nivolumab, pembrolizumab, and durvalumab) and chemotherapy (cisplatin, gemcitabine, and paclitaxel) was monitored in real-time with Cytotox Red staining in an Incucyte device, and the immunotumoroid response was compared to retrospective clinical drug responses. Results: Six of the 13 cases tested grew viable immunotumoroid models, with failed cases attributed to extensive tumor tissue necrosis or excess lymphocytes preventing spheroid formation. One successfully cultured case was excluded from the study due to low TIL infiltration (<5%) in the primary tumor sample. The five remaining models contained immune cells (CD4+ and CD8+ T cells, and macrophages), non-immune cells (fibroblasts), and tumor cells. Chemotherapy and ICI drugs were tested in immunotumoroids from 5 cases and compared to clinical outcomes where data was available. Four/five models showed cell killing in response to chemotherapy and two/five showed sensitivity to ICI. In three cases, the immunotumoroid model accurately predicted the patient's clinical response or non-response to ICIs or chemotherapy. Conclusion: Our immunotumoroid model replicated the multicellular nature of the tumor microenvironment sufficiently for preclinical ICI screening. This model could enable valuable insights into the complex interactions between cancer cells, the immune system, and the microenvironment. This is a feasibility study on a small number of cases, and additional studies with larger case numbers are required including correlation with clinical response.

Intratumoral Injection of Clostridium novyi-NT Spores in Patients with Treatment-refractory Advanced Solid Tumors.

PURPOSE: Intratumorally injected Clostridium novyi-NT (nontoxic; lacking the alpha toxin), an attenuated strain of C. novyi, replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation. PATIENTS AND METHODS: This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of C. novyi-NT across 6 dose cohorts (1 × 104 to 3 × 106 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose. RESULTS: Among 24 patients, a single intratumoral injection of C. novyi-NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses. The cohort 5 dose (1 × 106 spores) was defined as the maximum tolerated dose; DLTs were grade 4 sepsis (n = 2) and grade 4 gas gangrene (n = 1), all occurring in three patients with injected tumors >8 cm. Other treatment-related grade ≥3 toxicities included pathologic fracture (n = 1), limb abscess (n = 1), soft-tissue infection (n = 1), respiratory insufficiency (n = 1), and rash (n = 1), which occurred across four patients. Of 22 evaluable patients, nine (41%) had a decrease in size of the injected tumor and 19 (86%) had stable disease as the best overall response in injected and noninjected lesions combined. C. novyi-NT injection elicited a transient systemic cytokine response and enhanced systemic tumor-specific T-cell responses. CONCLUSIONS: Single intratumoral injection of C. novyi-NT is feasible. Toxicities can be significant but manageable. Signals of antitumor activity and the host immune response support additional studies of C. novyi-NT in humans.

The role of systems biology approaches in determining molecular signatures for the development of more effective vaccines.

INTRODUCTION: Emerging infectious diseases are a major threat to public health, and while vaccines have proven to be one of the most effective preventive measures for infectious diseases, we still do not have safe and effective vaccines against many human pathogens, and emerging diseases continually pose new threats. The purpose of this review is to discuss how the creation of vaccines for these new threats has been hindered by limitations in the current approach to vaccine development. Recent advances in high-throughput technologies have enabled scientists to apply systems biology approaches to collect and integrate increasingly large datasets that capture comprehensive biological changes induced by vaccines, and then decipher the complex immune response to those vaccines. AREAS COVERED: This review covers advances in these technologies and recent publications that describe systems biology approaches to understanding vaccine immune responses and to understanding the rational design of new vaccine candidates. EXPERT OPINION: Systems biology approaches to vaccine development provide novel information regarding both the immune response and the underlying mechanisms and can inform vaccine development.

Mast cells promote small bowel cancer in a tumor stage-specific and cytokine-dependent manner.

Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10-expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

p53 codon 72 polymorphism in basal cell carcinoma of the skin.

Basal cell carcinoma (BCC) is the most prevalent cancer in Iran. A common polymorphism at codon 72 of exon 4 of p53 tumor suppressor gene has been reported to be associated with increased inheritable susceptibility to several cancers. In the present study the frequency of p53 codon 72 polymorphism in 91 patients with BCC of skin, compared to 465 healthy normal individuals, was investigated. In total, there was no significant difference in the p53 genotypes between patients and controls. However, there was an apparent increase in the Arg/Arg genotype among those BCC patients who had a history of occupational sun exposure, compared to non-exposed patients (46.3% vs. 23.1%, P=0.11). A trend of increase in the frequency of Arg allele among sun-exposed patients was also observed (69.4% vs. 53.8%, P=0.07). Comparison of the genotype frequencies between sunexposed patients and normal controls confirmed the accumulation of Arg/Arg genotype in these patients (46.3% vs. 34.8%, P = 0.07). In addition, the frequency of Arg allele was significantly higher in sunexposed patients compared to controls (69.4% vs. 58.2%, P=0.03). Our results suggest that Arg allele at codon 72 of p53 gene might affect the risk of ultraviolet-induced basal cell carcinoma.

The promoter region (-800, -509) polymorphisms of transforming growth factor-beta1 (TGF-beta1) gene and recurrent spontaneous abortion.

Recurrent spontaneous abortion (RSA) is regarded as a common pregnancy complication in southern Iran. The exact causes of RSA are not yet known. Transforming growth factor-beta1 (TGF-beta1) is produced by T regulatory lymphocytes (Treg), which play an important role in the physiology of pregnancy. Several polymorphisms of the TGF-beta1 gene have been reported, some with important correlation with disease severity. In this investigation, the polymorphism of the TGF-beta1 gene at promoter region positions -800 (G/A) and -509 (C/T) was studied in 111 RSA and 110 normal female subjects from southern Iran by PCR-RFLP. Results indicated that at position -800 (G/A) polymorphism, 75.7% of RSA cases and 77.3% of normals were homozygote GG. In addition, 23.4% of cases and 22.7% of normal individuals were heterozygote AG. Only one of the patients appeared to be homozygote AA. None of the normal individuals were found to be homozygote AA at this position. In the case of the -509 (C/T) polymorphism, 38.7% of patients and 28.2% of controls were homozygote CC. While 40.6% of cases and 50.9% of normal individuals were heterozygote CT, 20.7% of RSA cases and 20.9% of controls were homozygote TT. The results indicate that there are no statistically significant differences of genotype distribution and allele frequency between RSA cases and controls at both polymorphic sites. In conclusion, the promoter region polymorphisms of TGF-beta1 at positions -800 (G/A) and -509 (C/T) may not be associated with RSA.

Cytotoxic T lymphocyte antigen-4 gene in breast cancer.

The exon 1 polymorphism (49A/G) of ctla-4 gene corresponds to an amino acid exchange (threonine to alanine) in the leader peptide of the expressed protein. There are reports concerning the higher level of G allele in subjects with various autoimmune diseases, which has resulted in the hypothesis that CTLA-4 may play a role in regulating self-tolerance by the immune system and in the pathogenesis of autoimmune disorders. This study was undertaken to investigate the correlation of exon 1 (49A/G) polymorphism in the ctla-4 gene and breast cancer. The ctla-4 49A/G polymorphism was studied in 197 women with primary breast cancer and 151 age/sex matched normal individuals. The results indicated a significant difference between frequency of ctla-4 genotypes in patients and controls. The frequency of GG genotype was significantly decreased in breast cancer patients compared to controls (4.6% v.s. 12.6%, P = 0.012). There was also a significant positive correlation between tumor size and the existence of AA genotype in patients (P = 0.016). In addition, a positive correlation between AA genotype and lymph node involvement was observed (P = 0.042). The observed decrease in the frequency of GG genotype in the breast cancer patients is contrary to the frequently reported increase of GG genotype in autoimmune diseases. In addition, the data implies that polymorphism of ctla-4 exon 1 contributes in tumor progression.