Post-irradiation intratumoral heterogeneity modulates response to immune checkpoint inhibition therapy in a murine melanoma model.

PURPOSE: The underlying mechanism for radiation as a potentiator of immune checkpoint inhibition (ICI) is unclear. We developed a novel murine model to investigate the effects of post-irradiation intratumoral heterogeneity (ITH) on response to ICI. EXPERIMENTAL DESIGN: Parental mouse melanoma B16F10 cells were irradiated in vitro (5Gy x 3 fractions), then an a priori determined number of resulting colonies were implanted in C57BL/6J immunocompetent mice creating syngeneic models of unirradiated (parental) and irradiated tumors with low (irradiated-L) and high (irradiated-H) ITH. Mice were treated with placebo, α-PD-L1, α-CTLA-4 or dual ICI. Murine tumors underwent whole exome sequencing (WES). Clinically correlated paired pre- and post-irradiation patient rectal adenocarcinoma samples underwent WES. RESULTS: Irradiated-L tumors showed increased tumor mutational burden (TMB) and a sustained decrease in ITH. Irradiated-L tumors were predicted to express five neoantigens with high variant allele frequency/clonal distribution. Mice with irradiated-L and irradiated-H versus parental B16F10 tumors demonstrated longer overall survival with dual ICI. Only mice with irradiated-L tumors experienced an overall survival benefit with single agent ICI. Clinically correlated rectal adenocarcinoma samples showed similarly increased TMB and decreased ITH following irradiation. CONCLUSIONS: Post-irradiation ITH modulates ICI response in a murine melanoma model. Irradiation may offer a mechanism to widen the therapeutic window of ICI.

Radiosensitivity of Breast Cancer Cells Is Dependent on the Organ Microenvironment.

Background: Distant metastasis is the leading risk factor of death in breast cancer patients, with lung and liver being commonly involved sites of distant seeding. Ongoing clinical trials are studying the benefit from additional local treatment to these metastatic sites with radiation therapy. However, little is known about the tissue-specific microenvironment and the modulating response to treatments due to limitations of traditional in vitro systems. By using biomatrix scaffolds (BMSs) to recreate the complex composition of extracellular matrices in normal organs, we chose to study the radiotherapy response with engineered breast cancer "metastases" in liver and lung organ-specific tissues. Methods: Liver and lung BMSs were prepared for tissue culture. Human breast cancer cell lines were passaged on normal tissue culture plates or tissue culture plates coated with Matrigel, liver BMSs, and lung BMSs. Clonogenic assays were performed to measure cell survival with varying doses of radiation. Reactive Oxygen Species (ROS) detection assay was used to measure ROS levels after 6 Gy irradiation to cancer cells. Results: The response of breast cell lines to varying doses of radiotherapy is affected by their in vitro acellular microenvironment. Breast cancer cells grown in liver BMSs were more radiosensitive than when grown in lung BMSs. ROS levels for breast cancer cells cultured in lung and liver BMSs were higher than that in plastic or in Matrigel plate cells, before and after radiotherapy, highlighting the interaction with surrounding tissue-specific growth factors and cytokines. ROSs in both lung and liver BMSs were significantly increased after radiotherapy delivery, suggesting these sites create prime environments for radiation-induced cell death. Conclusions: The therapeutic response of breast cancer metastases is dependent on the organ-specific microenvironment. The interaction between tissue microenvironment in these organs may identify sensitivity of therapeutic drug targets and radiation delivery for future studies.

PARP-1 genetic polymorphism associated with radiation sensitivity of non-small cell lung cancer.

About 70% of non-small cell lung cancer (NSCLC) patients require radiotherapy. However, due to the difference in radiation sensitivity, the treatment outcome may differ for the same pathology and choice of treatment. Poly (ADP-ribose) polymerase 1 (PARP-1) is a key gene responsible for DNA repair and is involved in base excision repair as well as repair of single strand break induced by ionizing radiation and oxidative damage. In order to investigate the relationship between PARP-1 gene polymorphism and radiation sensitivity in NSCLC, we collected 141 primary NSCLC patients undergoing three-dimensional conformal radiotherapy. For each case, the gross tumor volumes (GTV) before radiation and that after 40 Gy radiation were measured to calculate the tumor regression rate. TaqMan real-time polymerase chain reaction was performed to genotype the single-nucleotide polymorphisms (SNPs). Genotype frequencies for PARP-1 genotypes were 14.2% for C/C, 44.7% for C/G and 41.1% for G/G. The average tumor regression rate after 40 Gy radiation therapy was 35.1% ± 0.192. Tumor regression rate of mid-term RT of C/C genotype was 44.6% ± 0.170, which was higher than that of genotype C/G and G/G (32.4% ± 0.196 and 34.8% ± 0.188, respectively) with statistical significance (F = 3.169 p = 0.045). The higher tumor regression rate in patients with C/C genotype suggested that G allele was a protective factor against radiation therapy. Using the median tumor regression rate of 34%, we divided the entire cohort into two groups, and found that the frequency distribution of PARP-1 gene rs3219073 had significant difference between these two groups (p < 0.05). These results showed that PARP-1 gene polymorphism may affect patient radiation sensitivity and predict the efficacy of radiotherapy. It therefore presents an opportunity for developing new therapeutic targets to improve radiotherapy outcome.

miR­7/SP1/TP53BP1 axis may play a pivotal role in NSCLC radiosensitivity.

MicroRNA­7 (miR­7) has been identified as a tumor suppressor in non­small cell lung cancer (NSCLC) and a radiosensitivity regulator. Numerous studies have revealed that specific protein 1 (SP1) plays a critical role in the tumorigenesis of various types of cancers and regulates radiosensitivity and tumor suppressor p53­binding protein 1 (TP53BP1), which plays an essential role in DNA repair. However, it is not clear whether miR­7 has a regulatory effect on SP1 and TP53BP1 in NSCLC. In the present study it was revealed that miR­7 directly binds to the 3'UTR of SP1, thereby suppressing SP1 expression to regulate radiosensitivity. Overexpression of miR­7 and SP1 and knockdown of miR­7 and SP1 were performed using lentiviral transfection. Protein and mRNA abundance of SP1 and TP53BP1 were determined using western blotting and RT­qPCR, respectively, while miR­7 binding to SP1 was validated using a luciferase reporter assay. Biological function analysis indicated that miR­7 negatively regulated SP1 and inhibited cell proliferation, migration, and invasion when combined with radiation. It was also revealed that the expression of TP53BP1 was positively regulated by SP1 or negatively regulated by miR­7. In conclusion, SP1 was a target of miR­7, and the decreased expression of SP1 resulting from miR­7 overexpression in NSCLC was vital for improving radiosensitivity in NSCLC cells. Moreover, SP1 expression was detected in 95 paired NSCLC and adjacent normal tissues, and it was determined that SP1 was significantly upregulated in NSCLC tissues and that its upregulation was correlated with the degree of tissue differentiation. Thus, SP1 and/or miR­7 may be potential molecular targets in NSCLC radiotherapy.

RALY may cause an aggressive biological behavior and a dismal prognosis in non-small-cell lung cancer.

RALY is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP), an RNA-binding protein that plays a role in mRNA splicing and metabolism, may be involved in tumorigenesis and development. Some studies have shown that RALY plays a role in promoting cancer in a variety of tumors. However, the biological function and molecular mechanism of RALY in non-small cell lung cancer (NSCLC) remain unknown. TCGA databases were used to gather RALY expression data in NSCLC, the results indicate that RALY is highly expressed in cancer tissue of NSCLC patients. Then we demonstrated that RALY gene expression was notably upregulated in NSCLC tissue and cell lines (A549 and SK-MES-1), and was associated with lymph node metastasis (P = 0.007) and poorer overall survival in NSCLC patients. Subsequently, RALY in A549 and SK-MES-1 cells was knocked down by lentivirus to analyze the consequences of RALY on the biological behavior of NSCLC cell lines. Our results indicated that RALY knockdown impaired NSCLC cells proliferation, migration, and invasion, as well as arrested cells in G1 phase, and the reintroduction of RALY recused its biological phenotype. Furthermore, RALY knockdown down-regulated the expression levels of c-Myc, Cyclin D1, CDK4, MMP9, Rho A ,Rho C, N-cadherin and ß-catenin, and up-regulated the expression levels of P27, Rho B and E-cadherin. Therefore, targeting RALY could be a promising molecular target for NSCLC treatment.

Association between H19 SNP rs217727 and lung cancer risk in a Chinese population: a case control study.

BACKGROUND: H19 was the first long non-coding RNA (lncRNA) to be confirmed. Recently, studies have suggested that H19 may participate in lung cancer (LC) development and progression. This study assessed whether single nucleotide polymorphisms (SNPs) in H19 are associated with the risk of LC in a Chinese population. METHODS: A case-control study was performed, and H19 SNP rs217727 was analyzed in 555 lung cancer patients from two hospitals and 618 healthy controls to test the association between this SNP and the susceptibility to LC. RESULTS: The A/A homozygous genotype of rs217727 was significantly associated with an increased LC risk (odds ratio (OR) = 1.661, 95% confidence interval (CI) = 1.155 to 2.388, P = 0.006). Significant associations remained after stratification by smoking status (P < 0.001). Furthermore, the A/A genotype had a higher risk of LC than those of G/G in the squamous cell carcinoma (OR = 2.022, P = 0.004) and adenocarcinoma (OR = 1.606, P = 0.045) subgroups. CONCLUSIONS: The rs217727 SNP in lncRNA H19 was significantly associated with susceptibility to LC, particularly in squamous cell carcinoma and adenocarcinoma, and identified the homozygous A/A genotype as a risk factor for LC.

Developing a mouse model of acute bacterial rhinosinusitis.

The objective of this study is to establish a mouse model of acute bacterial rhinosinusitis. 179 healthy male BALB/c mice were divided into four groups in this randomized and controlled study. Sponge slivers impregnated with methicillin-resistant Staphylococcus aureus (MRSA COL) suspension were inserted into the right nasal cavities for group A; sponge slivers impregnated with sterile saline were inserted into the right nasal cavities for group B; group C mice were inoculated with MRSA COL suspension in right nasal cavities; group D was control group without any treatment. Mice were killed on days 1, 4, 7 and 14, respectively. Nasal lavage fluid was prepared for microbiological culture. Histological examinations of nasal specimens were performed to observe the severity of inflammatory reaction. Acute bacterial rhinosinusitis was induced in all group A mice. Less severe inflammation was seen in partial group B mice compared with that in group A mice (P ≤ 0.05). No inflammatory reaction was found in group C and D mice. In conclusion, a mouse model of acute bacterial rhinosinusitis has been developed successfully using an easier, less invasive and potentially more reversible technique than those used in previous studies.