Jerome Lacombe

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• Lacombe, J., Riou, O., Solassol, J., Mange, A., Ozsahin, M., & Azria, D. (2014).

  Susceptibilité individuelle et toxicité de la radiothérapie

  . In Les biomarqueurs moléculaires en oncologie. Springer, Paris. doi:10.1007/978-2-8178-0445-3_9
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  En 1901, six ans apres la decouverte des rayons X par Wilhelm Rontgen, Henri Becquerel et Pierre Curie publient une note officielle a l’Academie des sciences sur l’action physiologique des rayons du radium [1]. En reproduisant l’experience de Friedrich Giesel, Pierre Curie decrit les diverses rougeurs, brulures et plaies qu’a provoquees l’exposition, durant une dizaine d’heures, de chlorure de baryum radifere sur son avant-bras. Les symptomes decrits sont les memes que ceux de Marie Curie et d’Henri Becquerel, lorsque ces derniers transportent dans une poche de leur gilet un petit tube metallique scelle avec quelques centigrammes de cette meme matiere active. Ces diverses observations conduisent rapidement a la conclusion de l’existence d’un effet biologique des rayonnements ionisants sur les tissus.

Journals/Publications

• Jones, C. W., Overbey, E. G., Lacombe, J., Ecker, A. J., Meydan, C., Ryon, K., Tierney, B., Damle, N., MacKay, M., Afshin, E. E., Foox, J., Park, J., Nelson, T. M., Suhail Mohamad, M., Byhaqui, S. G., Aslam, B., Tali, U. A., Nisa, L., Menon, P. V., , Patel, C. O., et al. (2024). Molecular and physiological changes in the SpaceX Inspiration4 civilian crew. Nature, 632(8027), 1155-1164.
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  Human spaceflight has historically been managed by government agencies, such as in the NASA Twins Study, but new commercial spaceflight opportunities have opened spaceflight to a broader population. In 2021, the SpaceX Inspiration4 mission launched the first all-civilian crew to low Earth orbit, which included the youngest American astronaut (aged 29), new in-flight experimental technologies (handheld ultrasound imaging, smartwatch wearables and immune profiling), ocular alignment measurements and new protocols for in-depth, multi-omic molecular and cellular profiling. Here we report the primary findings from the 3-day spaceflight mission, which induced a broad range of physiological and stress responses, neurovestibular changes indexed by ocular misalignment, and altered neurocognitive functioning, some of which match those of long-term spaceflight, but almost all of which did not differ from baseline (pre-flight) after return to Earth. Overall, these preliminary civilian spaceflight data suggest that short-duration missions do not pose a significant health risk, and moreover present a rich opportunity to measure the earliest phases of adaptation to spaceflight in the human body at anatomical, cellular, physiological and cognitive levels. Finally, these methods and results lay the foundation for an open, rapidly expanding biomedical database for astronauts, which can inform countermeasure development for both private and government-sponsored space missions.
• Zhao, Z., Lacombe, J., Simon, L., Sanchez-Ballester, N. M., Khanishayan, A., Shaik, N., Case, K., Dugas, P. Y., Repellin, M., Lollo, G., Soulairol, I., Harris, A. F., Gordon, M., Begu, S., & Zenhausern, F. (2024). Physical, biochemical, and biological characterization of olive-derived lipid nanovesicles for drug delivery applications. Journal of nanobiotechnology, 22(1), 720.
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  Extracellular vesicles (EVs) have shown great promise as drug delivery system (DDS). However, their complex and costly production limit their development for clinical use. Interestingly, the plant kingdom can also produce EV-like nanovesicles that can easily be isolated and purified from a large quantity of raw material at a high yield. In this study, olive-derived nanovesicles (ODNVs) were isolated from raw fruits using serial centrifugations and their physical and biological features characterized to demonstrate their promising potential to be used as a DDS. Nanotracking particle analysis indicated an average size of 109.5 ± 3.0 nm and yield of 10 ODNVs/mL for the purest fraction. Microscopy imaging, membrane fluidity assay and lipidomics analysis showed the presence of a rich lipid bilayer that significantly varied between different sources of ODNVs but showed a distinct signature compared to human EVs. Moreover, ODNVs were enriched in PEN1 and TET8 compared to raw fruits, suggesting an extracellular origin. Interestingly, ODNVs size and yield stayed unchanged after exposure to high temperature (70 °C for 1 h), wide pH range (5-10), and 50-100 nm extrusion, demonstrating high resistance to physical and chemical stresses. This high resistance allowed ODNVs to stay stable in water at 4 °C for a month, or with the addition of 25 mM trehalose for long-term freezing storage. Finally, ODNVs were internalized by both 2D and 3D cell culture without triggering significant cytotoxicity and immunogenicity. Importantly, the anticancer drug doxorubicin (dox) could be loaded by passive incubation within ODNVs and dox-loaded ODNVs decreased cell viability by 90% compared to only 70% for free dox at the same concentration, indicating a higher efficiency of drug delivery by ODNVs. In addition, this high cytotoxicity effect of dox-loaded ODNVs was shown to be stable after a 2-week storage at 4 °C. Together, these findings suggested that ODNVs represent a promising candidate as drug nanocarrier for various DDS clinical applications, as demonstrated by their biocompatibility, high resistance to stress, good stability in harsh environment, and improvement of anticancer drug efficacy.
• Abend, M., Amundson, S. A., Badie, C., Brzoska, K., Kriehuber, R., Lacombe, J., Lopez-Riego, M., Lumniczky, K., Endesfelder, D., O'Brien, G., Doucha-Senf, S., Ghandhi, S. A., Hargitai, R., Kis, E., Lundholm, L., Oskamp, D., Ostheim, P., Schüle, S., Schwanke, D., , Shuryak, I., et al. (2023). RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay. Radiation research, 199(6), 598-615.
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  Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses 3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.
• Lacombe, J., Summers, A. J., Khanishayan, A., Khorsandian, Y., Hacey, I., Blackson, W., & Zenhausern, F. (2023). Paper-based vertical flow immunoassay for the point-of-care multiplex detection of radiation dosimetry genes. Cytogenetic and genome research.
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  In a nuclear or radiological incident, first responders must quickly and accurately measure radiation exposure among civilians, as medical countermeasures are radiation dose-dependent and time-sensitive. Although several approaches have been explored to measure absorbed radiation dose, there is an important need to develop point-of-care (POC) bioassay devices that can be used immediately to triage thousands of individuals potentially exposed to radiation.. Here we present a proof-of-concept study showing the use of a paper-based vertical flow immunoassay (VFI) to detect radiation dosimetry genes. Using labeled primers during amplification and a multiplex membrane, our results showed that the nucleic acid VFI can simultaneously detect two biodosimetry genes, CDKN1A and DDB2, as well as one housekeeping gene MRPS5. The assay demonstrated good linearity and precision with an inter- and intra-assay coefficient of variance < 20% and < 10%, respectively. Moreover, the assay showed its ability to discriminate non-irradiated controls (0 Gy) from irradiated samples (1 + 2 Gy) (p = 0.004). Interestingly, the gene combination also showed a dose-dependent response for 0, 1 and 2 Gy, similar to data obtained by real-time PCR benchmark. These preliminary results suggest that a VFI platform can be used to detect simultaneously multiple genes that can be then quantified, thus offering a new approach for a POC biodosimetry assay that could be rapidly deployed on-site to test a large population and help triage and medical management after radiological event.
• Port, M., Barquinero, J. F., Endesfelder, D., Moquet, J., Oestreicher, U., Terzoudi, G., Trompier, F., Vral, A., Abe, Y., Ainsbury, L., Alkebsi, L., Amundson, S. A., Badie, C., Baeyens, A., Balajee, A. S., Balázs, K., Barnard, S., Bassinet, C., Beaton-Green, L. A., , Beinke, C., et al. (2023). RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays. Radiation research, 199(6), 535-555.
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  Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses 2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.
• Harris, A. F., Lacombe, J., Sanchez-Ballester, N. M., Victor, S., Curran, K. A., Nordquist, A. R., Thomas, B., Gu, J., Veuthey, J. L., Soulairol, I., & Zenhausern, F. (2022). Decellularized Spinach Biomaterials Support Physiologically Relevant Mechanical Cyclic Strain and Prompt a Stretch-Induced Cellular Response. ACS applied bio materials, 5(12), 5682-5692.
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  Recently, decellularized plant biomaterials have been explored for their use as tissue engineered substitutes. Herein, we expanded upon the investigation of the mechanical properties of these materials to explore their elasticity as many anatomical areas of the body require biomechanical dynamism. We first constructed a device to secure the scaffold and induce a strain within the physiological range of the normal human adult lung during breathing (12-20 movements/min; 10-20% elongation). Results showed that decellularized spinach leaves can support cyclic strain for 24 h and displayed heterogeneous local strain values (7.76-15.88%) as well as a Poisson's ratio (0.12) similar to that of mammalian lungs (10.67-19.67%; 0.01), as opposed to an incompressible homogeneous standard polymer (such as PDMS (10.85-12.71%; 0.4)). Imaging and mechanical testing showed that the vegetal scaffold exhibited strain hardening but maintained its structural architecture and water retention capacity, suggesting an unaltered porosity. Interestingly, we also showed that cells seeded on the scaffold can also sense the mechanical strain as demonstrated by a nuclear reorientation perpendicular to strain direction (63.3° compared to 41.2° for nonstretched cells), a nuclear location of YAP and increased expression of YAP target genes, a high cytoplasmic calcium level, and an elevated expression level of collagen genes (COL1A1, COL3A1, COL4A1, and COL6A) with an increased collagen secretion at the protein level. Taken together, these data demonstrated that decellularized plant leaf tissues have an inherent elastic property similar to that found in the mammalian system to which cells can sense and respond.
• Lacombe, J., & Zenhausern, F. (2022). Effect of mechanical forces on cellular response to radiation. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 176, 187-198.
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  While the cellular interactions and biochemical signaling has been investigated for long and showed to play a major role in the cell's fate, it is now also evident that mechanical forces continuously applied to the cells in their microenvironment are as important for tissue homeostasis. Mechanical cues are emerging as key regulators of cellular drug response and we aimed to demonstrate in this review that such effects should also be considered vital for the cellular response to radiation. In order to explore the mechanobiology of the radiation response, we reviewed the main mechanoreceptors and transducers, including integrin-mediated adhesion, YAP/TAZ pathways, Wnt/β-catenin signaling, ion channels and G protein-coupled receptors and showed their implication in the modulation of cellular radiosensitivity. We then discussed the current studies that investigated a direct effect of mechanical stress, including extracellular matrix stiffness, shear stress and mechanical strain, on radiation response of cancer and normal cells and showed through preliminary results that such stress effectively can alter cell response after irradiation. However, we also highlighted the limitations of these studies and emphasized some of the contradictory data, demonstrating that the effect of mechanical cues could involve complex interactions and potential crosstalk with numerous cellular processes also affected by irradiation. Overall, mechanical forces alter radiation response and although additional studies are required to deeply understand the underlying mechanisms, these effects should not be neglected in radiation research as they could reveal new fundamental knowledge for predicting radiosensitivity or understanding resistance to radiotherapy.
• Lacombe, J., Muccio, M., & Zenhausern, F. (2022).

  Abstract 5696: Effect of extracellular matrix stiffness on cancer cells radiation response

  . Cancer Research, 82(12_Supplement), 5696-5696. doi:10.1158/1538-7445.am2022-5696
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  Abstract Over the past decade, it has become clear that the mechanical cues of extracellular matrix (ECM) are a major regulator of the cancer biology. In particular, ECM stiffness has shown an important role in chemoresistance modulation. However, despite the central role of radiotherapy in cancer management, little is known about the effect of ECM stiffness on cancer cells radiation response. In this study, we used polyacrylamide collagen-coated hydrogel model to reproduce a soft (0.7 kPa) and a stiff (70 kPa) matrix. The model was validated by atomic force microscopy force spectroscopy and biological analyses through cellular morphology, proliferation and YAP/TAZ mechanotransduction pathway that all confirmed the accuracy of our model. Then, lung cancer cells seeded on both soft and stiff hydrogels were irradiated, and the level of DNA damages were compared between the two conditions. First, alkaline comet assay revealed the total amount of DNA damages were higher in cells grown on a rigid ECM at 1 h post-irradiation than cells seeded on a soft matrix. Second, γH2AX/53BP1 foci assay showed that the number of DNA double strand breaks were also higher in cells grown on a rigid environment. However, the level of DNA damage was identical at 24 h post-irradiation for both stiffness, suggesting that a more efficient DNA damage repair response could take place for cells seeded in stiff matrix. In order to explain these observations, analyses on chromatin structure and nucleus shape were then performed. Data showed that cells cultured on stiff matrix displayed a larger nucleus, less condensed chromatin, and a decreased expression level of class I HDACs genes. Moreover, it was found that the level of total reactive oxygen species (ROS) and superoxide ion was also higher in cells grown on a rigid environment 24 h post-irradiation. Altogether these data suggest that a high stiffness may increase early DNA damages after irradiation, partially by remodeling chromatin that could expose DNA to radiation-induced genotoxic agents such as ROS. Additional investigations are required to understand better the mechanisms that could explain such differences, but these preliminary data highlighted the potential role of mechanical stress in cellular radiation response and paved the way to new therapeutic target for radiotherapy management. Citation Format: Jerome Lacombe, Melinda Muccio, Frederic Zenhausern. Effect of extracellular matrix stiffness on cancer cells radiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5696.
• Lacombe, J., Soldevila, M., & Zenhausern, F. (2022). From organ-on-chip to body-on-chip: The next generation of microfluidics platforms for in vitro drug efficacy and toxicity testing. Progress in molecular biology and translational science, 187(1), 41-91.
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  The high failure rate in drug development is often attributed to the lack of accurate pre-clinical models that may lead to false discoveries and inconclusive data when the compounds are eventually tested in clinical phase. With the evolution of cell culture technologies, drug testing systems have widely improved, and today, with the emergence of microfluidics devices, drug screening seems to be at the dawn of an important revolution. An organ-on-chip allows the culture of living cells in continuously perfused microchambers to reproduce physiological functions of a particular tissue or organ. The advantages of such systems are not only their ability to recapitulate the complex biochemical interactions between different human cell types but also to incorporate physical forces, including shear stress and mechanical stretching or compression. To improve this model, and to reproduce the absorption, distribution, metabolism, and elimination process of an exogenous compound, organ-on-chips can even be linked fluidically to mimic physiological interactions between different organs, leading to the development of body-on-chips. Although these technologies are still at a young age and need to address a certain number of limitations, they already demonstrated their relevance to study the effect of drugs or toxins on organs, displaying a similar response to what is observed in vivo. The purpose of this review is to present the evolution from organ-on-chip to body-on-chip, examine their current use for drug testing and discuss their advantages and future challenges they will face in order to become an essential pillar of pharmaceutical research.
• Bennet, D., Harris, A. F., Lacombe, J., Brooks, C., Bionda, N., Strickland, A. D., Eisenhut, T., & Zenhausern, F. (2021). Evaluation of supercritical CO sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2. The Science of the total environment, 780, 146519.
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  The purpose of this research is to evaluate the supercritical carbon dioxide (scCO) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a "worst-case" regarding challenged PPE. The inactivation of the microbes by scCO sterilization with NovaKill or HO sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.
• Drenner, K., Halder, T. G., Kaadige, M., Lacombe, J., Ng, S., Sharma, S., Soldi, R., Thode, T., Weston, A., & del Villar, R. R. (2021).

  Abstract 3068: Everolimus mediated Radioprotection on surrounding healthy tissue in glioblastoma microbrain models

  . Cancer Research, 81(13_Supplement), 3068-3068. doi:10.1158/1538-7445.am2021-3068
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  Abstract Background: After surgical resection, ionizing radiation alone or in combination with chemotherapy is the main treatment modality for brain tumors including glioblastoma (GBM). Although the effect of radiation-induced (RI) damage improves the patient survival ratio, injury to the neural stem and progenitor cell (NSPC) compartments and damage to NSPC populations is hypothesized to be central to the pathogenesis of RI cognitive decline in the patients. Studies on sensitivity of neural cell populations to radiation have shown that mature neurons and astrocytes have substantial radio resistance; in contrast, NSPC are highly sensitive to radiation, as exposure induces DNA double-strand breaks that when unrepaired can lead to cell death. Therefore, protection of NPCs is an important countermeasure against radiotherapy in normal tissues. Rapamycin, a small molecule inhibitor of mammalian target of rapamycin (mTOR), has been shown to exert a profoundly protective effect on epithelial and hematopoietic stem cells upon exposure to radiation therapy. Recently, several pharmacokinetically improved rapamycin analogs have secured FDA approval. In this study we investigate the potential of Everolimus as radio-protector for NSPCs in a GBM environment. Materials and Methods: NSPCs generated from healthy donors and GBM cells were treated with different doses of Everolimus 24 h before and after a single dose 4 Gy irradiation. Survival was monitored using Cell Titer Glo assay. Western Blots, qPCR and immunofluorescence were used to assess mTOR pathway activation and NPC differentiation in 2D and 3D culture. Co-culture of NSPCs and GFP-tagged GBM cells were treated with Everolimus and irradiated with subsequent followup by confocal imaging to check the relative survival of these cells. Results: Low doses of Everolimus restored survival and proliferation of irradiated NSPCs, while GBM cells remained radiosensitive. qPCR analysis of Nestin, SOX2 and FOXG1 revealed that NSPCs maintained their stemness after treatment. MAP2 and βIII tubulin analysis demonstrated that irradiated NSPCs were able to differentiate to neurons upon treatment. Everolimus treatment also reduced γH2A and phospho-PP2A levels and promoted phosphorylation of Akt suggesting that mTOR inhibition with Everolimus could activate DNA repair in irradiated NPCs. Conclusions: Our findings suggest that pretreatment with low-dose Everolimus may protect NSPCs from radiation induced injuries after radiation therapy, while maintaining radiation sensitivity to GBM cells. This study suggests low-dose Everolimus as a potential radio-protector for clinical treatment of glioblastoma and other brain malignancies. Citation Format: Tithi Ghosh Halder, Ryan Rodriguez del Villar, Jerome Lacombe, Kevin Drenner, Serina Ng, Trason Thode, Alexis Weston, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. Everolimus mediated Radioprotection on surrounding healthy tissue in glioblastoma microbrain models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3068.
• Harris, A. F., Lacombe, J., & Zenhausern, F. (2021). The Emerging Role of Decellularized Plant-Based Scaffolds as a New Biomaterial. International journal of molecular sciences, 22(22).
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  The decellularization of plant-based biomaterials to generate tissue-engineered substitutes or in vitro cellular models has significantly increased in recent years. These vegetal tissues can be sourced from plant leaves and stems or fruits and vegetables, making them a low-cost, accessible, and sustainable resource from which to generate three-dimensional scaffolds. Each construct is distinct, representing a wide range of architectural and mechanical properties as well as innate vasculature networks. Based on the rapid rise in interest, this review aims to detail the current state of the art and presents the future challenges and perspectives of these unique biomaterials. First, we consider the different existing decellularization techniques, including chemical, detergent-free, enzymatic, and supercritical fluid approaches that are used to generate such scaffolds and examine how these protocols can be selected based on plant cellularity. We next examine strategies for cell seeding onto the plant-derived constructs and the importance of the different functionalization methods used to assist in cell adhesion and promote cell viability. Finally, we discuss how their structural features, such as inherent vasculature, porosity, morphology, and mechanical properties (i.e., stiffness, elasticity, etc.) position plant-based scaffolds as a unique biomaterial and drive their use for specific downstream applications. The main challenges in the field are presented throughout the discussion, and future directions are proposed to help improve the development and use of vegetal constructs in biomedical research.
• Harris, A. F., Lacombe, J., Liyanage, S., Han, M. Y., Wallace, E., Karsunky, S., Abidi, N., & Zenhausern, F. (2021). Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds. Scientific reports, 11(1), 3643.
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  The use of plant-based biomaterials for tissue engineering has recently generated interest as plant decellularization produces biocompatible scaffolds which can be repopulated with human cells. The predominant approach for vegetal decellularization remains serial chemical processing. However, this technique is time-consuming and requires harsh compounds which damage the resulting scaffolds. The current study presents an alternative solution using supercritical carbon dioxide (scCO). Protocols testing various solvents were assessed and results found that scCO in combination with 2% peracetic acid decellularized plant material in less than 4 h, while preserving plant microarchitecture and branching vascular network. The biophysical and biochemical cues of the scCO decellularized spinach leaf scaffolds were then compared to chemically generated scaffolds. Data showed that the scaffolds had a similar Young's modulus, suggesting identical stiffness, and revealed that they contained the same elements, yet displayed disparate biochemical signatures as assessed by Fourier-transform infrared spectroscopy (FTIR). Finally, human fibroblast cells seeded on the spinach leaf surface were attached and alive after 14 days, demonstrating the biocompatibility of the scCO decellularized scaffolds. Thus, scCO was found to be an efficient method for plant material decellularization, scaffold structure preservation and recellularization with human cells, while performed in less time (36 h) than the standard chemical approach (170 h).
• Clements, T., Devadhasan, J. P., Gu, J., Kuehl, P., Lacombe, J., Stoudemire, J., & Zenhausern, F. (2020).

  Abstract: Automation of the paper-based vertical flow platform for radiation biodosimetry during deep space mission

  . 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2020.
• Cosson, P., Lacombe, J., & Zenhausern, F. (2020).

  AI334, AQ806, AR222, AR249, AS274, AS708, RB572, RB574 and RB596 antibodies recognize SARS-CoV-2 viral particles by dot blot

  . Antibody Reports, 3(4), e273. doi:10.24450/journals/abrep.2020.e273
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  The recombinant antibodies AI334, AQ806, AR222, AR249, AS274, AS708, RB572, RB574 and RB596 detect inactivated SARS-CoV-2 viral particles by dot blot.
• Lacombe, J., Harris, A. F., Zenhausern, R., Karsunsky, S., & Zenhausern, F. (2020). Plant-Based Scaffolds Modify Cellular Response to Drug and Radiation Exposure Compared to Standard Cell Culture Models. Frontiers in bioengineering and biotechnology, 8, 932.
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  Plant-based scaffolds present many advantages over a variety of biomaterials. Recent studies explored their potential to be repopulated with human cells and thus highlight a growing interest for their use in tissue engineering or for biomedical applications. However, it is still unclear if these plant-based scaffolds can modify cell phenotype or affect cellular response to external stimuli. Here, we report the characterization of the mechano-regulation of melanoma SK-MEL-28 and prostate PC3 cells seeded on decellularized spinach leaves scaffolds, compared to cells deposited on standard rigid cell culture substrate, as well as their response to drug and radiation treatment. The results showed that YAP/TAZ signaling was downregulated, cellular morphology altered and proliferation rate decreased when cells were cultured on leaf scaffold. Interestingly, cell culture on vegetal scaffold also affected cellular response to external stress. Thus, SK-MEL-28 cells phenotype is modified leading to a decrease in MITF activity and drug resistance, while PC3 cells showed altered gene expression and radiation response. These findings shed lights on the decellularization of vegetal materials to provide substrates that can be repopulated with human cells to better reproduce a soft tissue microenvironment. However, these complex scaffolds mediate changes in cell behavior and in order to exploit the capability of matching physical properties of the various plant scaffolds to diverse physiological functionalities of cells and human tissue constructs, additional studies are required to better characterize physical and biochemical cell-substrate interactions.
• Cretignier, T., Cuendet, M., Gunatilaka, L., Lacombe, J., Meli, L., Veuthey, J., & Zenhausern, F. (2019).

  Abstract 2932: Withanolide D enhances radiosensitivity of human cancer cells to X-rays radiation

  . Tumor Biology, 79(13_Supplement), 2932-2932. doi:10.1158/1538-7445.am2019-2932
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  Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, with about half of cancer patients receiving RT during their treatment. However, there are still a lot of challenges to improve its efficiency by minimizing normal tissue toxicity while maximizing tumor control. In this perspective, radiosensitizers are a promising approach in order to create a better tumor response to ionizing radiation (IR) while allowing for better dose modulation. Withanolides are natural steroidal lactones found in the plant Withania somnifera and already known for their numerous biological effects, in particular an antitumor activity due to induction of ROS production, cell cycle arrest or cytoskeleton destabilization. Hence, we tested in this study our hypothesis that withanolide D (WD), a compound with an important antitumor effect, could also act as a radiosensitizer. Clonogenic assays showed that 1-hour WD pretreatment (0.7µM) before IR decreased surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieves its radiosensitizing effect, we then assessed whether WD could promote radiation-induced DNA damages and inhibit double-strand breaks (DSBs) repair in SKOV3 cells. Comet and γH2AX/53BP1 foci formation assays confirmed that DSBs are higher from 1 hour up to 24 hours after 2Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induces the persistence of radiation-induced DNA damages. We then performed immunoblotting to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PK, ATM and their phosphorylated forms appeared to be inhibited at 24 hours post-irradiation in WD-treated samples. XRCC4 expression is also decreased while RAD51 expression does not change compared to vehicle-treated cells suggesting that only non-homologous end joining pathways is altered by WD. In order to assess the consequence of such inhibition, we then investigated cell death, and especially delayed death, referred to as mitotic catastrophe (MC), which was described as the main form of epithelial cell death induced by IR. MC is induced after IR and but is predominant in WD-treated samples as showed by the few numbers of cells pursing into anaphase. Interestingly, our results showed that cells preferentially undergo necrosis-like death after MC instead of apoptosis. This can be explained by the p53 mutated profile of SKOV3 in addition to the WD-induced ATM inhibition. Together, these data demonstrate that WD is a promising radiosensitizer candidate for RT and additional studies are required to investigate its effect in more relevant clinical models. Citation Format: Jerome Lacombe, Titouan Cretignier, Laetitia Meli, Jean-Luc Veuthey, Leslie Gunatilaka, Muriel Cuendet, Frederic Zenhausern. Withanolide D enhances radiosensitivity of human cancer cells to X-rays radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2932.
• Lacombe, J., Zenhausern, R. S., & Zenhausern, F. (2018).

  Abstract 1316: Vegetal scaffold as radiobiology model to study radiation cancer response

  . Cancer Research, 78(13_Supplement), 1316-1316. doi:10.1158/1538-7445.am2018-1316
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  Current in vitro tumor models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found in vivo. The use of decellularized plant structures recellularized with human cells, aims to overcome these issues by taking advantage of their natural 3D structure. By using this approach on spinach leaves as a 3D scaffold, we have developed a new model that may be used as a new tumor model for radiobiology research. Spinach leaves were decellularized following serial chemical treatments with hexanes, SDS, Triton-X100 and bleach. In order to characterize the efficiency of the decellularization process, the rigidity of the leaves was assessed by Atomic Force Microscope (AFM) and DNA and protein quantification. Human prostate (PC3) and breast (MCF7) cancer cells were then seeded onto leaf. Seeding efficiency was assessed by optical microscopy and viability and proliferation ability were tested by MTT assay. In order to evaluate if the cells were biologically active, we then assessed radiation response. Extra cell-seeded leaves were irradiated and the expression of radiation-responsive genes were assessed in MCF7 cells. Additionally, DNA damage levels in PC3 cells were evaluated by γ-H2AX foci measurement using fluorescence microscopy. The decellularization process was successful, showing a protein content of 0.31 μg/mg tissue compared to the fresh leaf at 14.4 μg/mg tissue. The DNA quantity was similarly disparate between fresh and decellularized leaves. Microscopy showed that PC3 and MCF7 cells were well attached to the decellularized leaf surface after 24h incubation. Mechanical testing with AFM confirmed attachment by measuring Young9s modulus values of 2.81, 88 and 197 MPa for decellularized, recellularized and fresh leaves respectively. Viability assays confirmed that cells were alive and able to proliferate. The gene expression assay showed changes in expression levels between 2D cell culture and cells seeded on leaves both at basal state and after 5Gy-irradiation in MCF7 cells. Finally, γ-H2AX immunofluorescent imaging showed DNA damage repairs are induced 1 hour after 5Gy of X-ray irradiation in PC3 cells and are effective up to 24h. Plant can be decellularized in order to create a 3D scaffold that may act as a support for cell seeding. Interestingly, radiation response can be measured on this new model and even show significant difference with standard 2D cell culture. Together, these results suggest that this approach is a new promising 3D cellular model for radiation research. However, additional studies are required to compare this model with in vivo response in order to clearly assess if this model is more suitable to mimick in vivo tumor/microenvironment than 2D standard model. Citation Format: Jerome Lacombe, Ryan Zenhausern, Frederic Zenhausern. Vegetal scaffold as radiobiology model to study radiation cancer response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1316.
• Lacombe, J., Solassol, J., Ozsahin, M., Coelho, M., Azria, D., & Pouget, J. P. (2012).

  OC-32: Quantitative Proteomic Approach to Identify Hypersensitive Patients to Curative Intent Radiotherapy

  . Radiotherapy and Oncology, 104(104), 31. doi:10.1016/s0167-8140(15)34586-2

Presentations

• Lacombe, J., Khanishayan, A., Summers, A. J., Amundson, S., Turner, H., & Zenhausern, F. (2023, September).

  Development of a point-of-care bioassay to detect radiation dosimetry blood biomarkers

  . 17th International Congress for Radiation Research. Montreal, Quebec, Canada.

Poster Presentations

• Summers, A. J., Devadhasan, J. P., Khanishayan, A., Lacombe, J., Gu, J., & Zenhausern, F. (2023, October). Gold Nanostar-Based Vertical Flow Immunoassay for Detection of Biothreat Agents. BMES Annual Meeting. Seattle, WA.