There are many challenges to live C. elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluorescence without the use of microfluidic technologies. The methods present here use common microscopy consumables and equipment found in many imaging core facilities and can be easily adapted to fit on multiple microscopy systems.
Radiotherapy is a commonly used cancer treatment; however, patients with high serum squamous cell carcinoma antigen (SCCA1/SERPINB3) are associated with resistance and poor prognosis. Despite being a strong clinical biomarker, the modulation of SERPINB3 in tumor immunity is poorly understood. We investigated the microenvironment of SERPINB3 high tumors through RNAseq of primary cervix tumors and found that SERPINB3 was positively correlated with CXCL1/8, S100A8/A9 and myeloid cell infiltration. Induction of SERPINB3 in vitro resulted in increased CXCL1/8 and S100A8/A9 production, and supernatants from SERPINB3-expressing cultures attracted monocytes and MDSCs. In murine tumors, the orthologue mSerpinB3a promoted MDSC, TAM, and M2 macrophage infiltration contributing to an immunosuppressive phenotype, which was further augmented upon radiation. Radiation-enhanced T cell response was muted in SERPINB3 tumors, whereas Treg expansion was observed. A STAT-dependent mechanism was implicated, whereby inhibiting STAT signaling with ruxolitinib abrogated suppressive chemokine production. Patients with elevated pre-treatment serum SCCA and high pSTAT3 had increased intratumoral CD11b+ myeloid cell compared to patients with low SCCA and pSTAT3 cohort that had overall improved cancer specific survival after radiotherapy. These findings provide a preclinical rationale for targeting STAT signaling in tumors with high SERPINB3 to counteract the immunosuppressive microenvironment and improve response to radiation.
The purpose of this study was to determine if squamous cell carcinoma antigen (SCCA), also known as SERPINB3, protects cervical cancer cells from ionizing radiation (IR)-induced death, and to determine the mechanism(s) of IR-induced cell death with or without SERPINB3. Cervical cancer remains a leading cause of cancer death in women worldwide despite advances in screening, vaccination and treatment. Recurrence after definitive chemoradiation occurs in up to 30-50% of patients with locally advanced disease. We and others have demonstrated that elevated serum SCCA portends poor prognosis in cervical cancer. In addition, CRISPR-Cas9-mediated knock out of SERPINB3 significantly sensitizes cervical tumor cells to IR in vitro. We hypothesize that SERPINB3 promotes radioresistance by protecting cells against lysosome damage and lysosomal mediated necrosis. Two cervical cancer cell lines with high SERPINB3 expression were edited using CRISPR-Cas9 technology at the SERPINB3 locus resulting in knock out (KO). B3-KO cells were significantly more radiation sensitive compared to control cells at all doses and time points evaluated. Cell death morphology in the B3-KO cells was necrotic, with large cytoplasmic single membraned vesicles, many of which were ruptured, consistent with that seen in lysosome-mediated necrosis. Live-cell time-lapse imaging showed loss of lysosome integrity in the hours leading up to cell death (propidium iodide nuclear staining). Western blot analysis showed low levels of caspase-3 and caspase-7 cleavage only at high doses and late time points after IR, with no evidence of phospho-MLKL or phospo-RIPK3 (markers of necroptosis), or gasdermin-D cleavage (marker of pyroptosis). Necrostatin, ferrostatin, liproxstatin and YVAD-Cho had little to no effect on cell death following IR, while pan-caspase inhibitors decreased IR-induced cell death in both control and B3-KO cells, and E64D decreased IR-induced cell death in B3-KO cells to a greater degree than control cells. Additionally, cervical tumor cell lines that had no detectable expression of SERPINB3 were engineered to expressed high levels of SERPINB3 (B3-wt) or SERPINB3 containing the P14 mutation A351R (B3-P14m), which lacks protease inhibitory function. B3-wt expressing cells displayed increased radiation resistance in clonogenic survival assays and mouse tumor models. Growth rate and radiation response of B3-p14m expressing tumors was similar to vector control. These data suggest that SERPINB3 is an important mediator of radiation response in cervical cancer and protects cells by inhibiting cysteine protease-dependent cell death, likely via lysosome-mediated necrosis. These findings support SERPINB3 as a potential target for novel radiosensitizing therapies. Citation Format: Songyan Wang, Clifford Luke, Victoria Shi, Perry Grigsby, Gary Silverman, Stephanie Markovina. SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2426.
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