Investigating Radiosensitivity of HPV-positive and HPV-negative Head and Neck Squamous Cell Carcinoma Spheroids by PARP Inhibition

Head and neck squamous cell carcinoma (HNSCC) is the sixth most-common cancer over the world, and it remains a significant public health concern in the UK. A number of studies have indicated HPV type-16 infection is one of the main risk factors related with nearly 50% HNSCC cases. Interestingly, the survival rate of HPV-positive HNSCC patients is shown to be higher than HPV-negative status, due to an improved prognosis and better response to chemo- and/or radiotherapeutic strategies. Previously published studies in our group have demonstrated the increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and the efficiency of DNA double strand break (DSB) repair. Therefore in this project, I demonstrate that HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) spheroids show higher radiosensitivity compared to HPV-negative OPSCC spheroids, due to the defective efficacy of DNA DSB repair. Subsequently, PARP inhibitors Olaparib and Talazoparib presented significant enhancement of radio-sensitisation on HPV-negative HNSCC spheroids. Moreover, HNSCC spheroid growth was more effectively suppressed by proton beam therapy in combination these PARP inhibitors. This research suggested that the combination of clinical PARPi and proton irradiation can become a potent treatment for HPV-negative HNSCC patients in the future

Effectiveness of PARP inhibition in enhancing the radiosensitivity of 3D spheroids of head and neck squamous cell carcinoma.

A critical risk factor for head and neck squamous cell carcinoma (HNSCC), particularly of the oropharynx, and the response to radiotherapy is human papillomavirus (HPV) type-16/18 infection. Specifically, HPV-positive HNSCC display increased radiosensitivity and improved outcomes, which has been linked with defective signalling and repair of DNA double-strand breaks (DSBs). This differential response to radiotherapy has been recapitulated in vitro using cell lines, although studies utilising appropriate 3D models that are more reflective of the original tumour are scarce. Furthermore, strategies to enhance the sensitivity of relatively radioresistant HPV-negative HNSCC to radiotherapy are still required. We have analysed the comparative response of in vitro 3D spheroid models of oropharyngeal squamous cell carcinoma to x-ray (photon) irradiation and provide further evidence that HPV-positive cells, in this case now grown as spheroids, show greater inherent radiosensitivity compared to HPV-negative spheroids due to defective DSB repair. We subsequently analysed these and an expanded number of spheroid models, with a particular focus on relatively radioresistant HPV-negative HNSCC, for impact of poly(ADP-ribose) polymerase (PARP) inhibitors (olaparib and talazoparib) in significantly inhibiting spheroid growth in response to photons but also proton beam therapy. We demonstrate that in general, PARP inhibition can further radiosensitise particularly HPV-negative HNSCC spheroids to photons and protons leading to significant growth suppression. The degree of enhanced radiosensitivity was observed to be dependent on the model and on the tumour site (oropharynx, larynx, salivary gland, or hypopharynx) from which the cells were derived. We also provide evidence suggesting that PARP inhibitor effectiveness relates to homologous recombination repair proficiency. Interestingly though, we observed significantly enhanced effectiveness of talazoparib versus olaparib specifically in response to proton irradiation. Nevertheless, our data generally support that PARP inhibition in combination with radiotherapy (photons and protons) should be considered further as an effective treatment for HNSCC, particularly for relatively radioresistant HPV-negative tumours

Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA) which vary in size and shape were used. Our results show that several polymers can be used to extract membrane proteins comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular weight (7.5-10 kDa) is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification SMA 2000 was found to be the best choice for yield, purity and function. However the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications

The radiobiology of HPV-positive and HPV-negative head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with reported incidences of ~800 000 cases each year. One of the critical determinants in patient response to radiotherapy, particularly for oropharyngeal cancers, is human papillomavirus (HPV) status where HPV-positive patients display improved survival rates and outcomes particularly because of increased responsiveness to radiotherapy. The increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and repair of DNA double-strand breaks. Therefore, strategies to further radiosensitise HPV-positive HNSCC, but also radioresistant HPV-negative HNSCC, have focussed on targeting key DNA repair proteins including PARP, DNA-Pk, ATM and ATR. However, inhibitors against CHK1 and WEE1 involved in cell-cycle checkpoint activation have also been investigated as targets for radiosensitisation in HNSCC. These studies, largely conducted using established HNSCC cell lines in vitro, have demonstrated variability in the response dependent on the specific inhibitors and cell models utilised. However, promising results are evident targeting specifically PARP, DNA-Pk, ATR and CHK1 in synergising with radiation in HNSCC cell killing. Nevertheless, these preclinical studies require further expansion and investigation for translational opportunities for the effective treatment of HNSCC in combination with radiotherapy

Functional Composition Changes of a Subtropical Monsoon Evergreen Broad-Leaved Forest Under Environmental Change

Long-term studies have revealed that forest species composition was shifting under environment change and disturbance induced by loss of large trees. Yet, few studies explicitly analyzed their impacts on composition concurrently. To learn more about impacts of environment change and disturbance on driving forest community, we investigated shifts in functional composition over past 24 years in an old-growth subtropical forest in southern China. We analyzed nine traits that are mainly related to leaf nutrients, photosynthetic capacity, hydraulic conductivity, and drought tolerance of plants and examined hypotheses: (1) The functional composition change over time was directional instead of random fluctuation, (2) drought-tolerant species increased their abundance under soil dryness, (3) both environmental change and disturbance related to changes of functional composition significantly, and (4) initial trait values of quadrats strongly influenced their subsequent change rates in quadrat level (10 × 10 m). We found that species composition had shifted to favor species with high leaf nutrient content, high photosynthesis rate, high hydraulic conductivity, low water-use efficiency, and high drought tolerance traits, which was due to soil dryness and disturbance. These two factors explained 47–58% of quadrats’ trait value changes together. Considering rapidly increasing stem density, this pattern may indicate ecological processes of which disturbance provided numerous recruits of resource-acquisition strategy species and soil dryness conducted a selecting effect on shaping composition in the forest. Additionally, quadrats with initial trait values at the far end of change direction shifted faster in three traits, which also indicated that functional composition changes in quadrats were directional and homogenized. Our results implied that environment change and accompanied disturbance events possibly drove species composition change along a different trajectory in the subtropical forest that experienced high climatic variability

Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR

Background Some COVID-19 cases test positive again for SARS-CoV-2 RNA following negative test results and discharge, raising questions about the meaning of virus detection. Better characterization of re-positive cases is urgently needed. Methods Clinical data were obtained through Guangdong's COVID-19 surveillance network. Neutralization antibody titre was determined using microneutralization assays. Potential infectivity of clinical samples was evaluated by cell inoculation. SARS-CoV-2 RNA was detected using three different RT-PCR kits and multiplex PCR with nanopore sequencing. Findings Among 619 discharged COVID-19 cases, 87 re-tested as SARS-CoV-2 positive in circumstances of social isolation. All re-positive cases had mild or moderate symptoms at initial diagnosis and were younger on average (median, 28). Re-positive cases (n = 59) exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases (n = 218) tested here. No infectious strain could be obtained by culture and no full-length viral genomes could be sequenced from re-positive cases. Interpretation Re-positive SARS-CoV-2 cases do not appear to be caused by active reinfection and were identified in ~14% of discharged cases. A robust NAb response and potential virus genome degradation were detected in almost all re-positive cases, suggesting a substantially lower transmission risk, especially through respiratory routes.ISSN:2352-396