Biomphalaria glabrata is an intermediate host of Schistosoma mansoni, the agent of human intestinal schistosomiasis. However, much is to be discovered about its innate immune system that appears as a complex black box, in which the immune cells  play a major role in both cellular and humoral response towards pathogens. Until now, hemocyte classification has been based exclusively on cell morphology and ultrastructural description and depending on the authors considered from 2 to 5 hemocyte populations have been described. In this study, we proposed to evaluate the hemocyte heterogeneity at the transcriptomic level. To accomplish this objective, we used single cell RNA sequencing (scRNAseq) technology coupled to a droplet-based system to separate hemocytes and analyze their transcriptome at a unique cell level in naive Biomphalaria glabrata snails. We were able to demonstrate the presence of 7 hemocyte transcriptomic populations defined by the expression of specific marker genes. As a result, scRNAseq approach showed a high heterogeneity within hemocytes, but provides a detailed description of the different hemocyte transcriptomic populations in B. glabrata supported by distinct cellular functions and lineage trajectory. As a main result, scRNAseq revealed the 3 main population as a super-group of hemocyte diversity but, on the contrary, a great hemocytes plasticity with a probable capacity of hemocytes to engage to different activation pathways. This work opens a new field of research to understand the role of hemocytes particularly in response to pathogens, and towards S. mansoni parasites.The freshwater snail  In invertebrates, innate immune response is mainly carried out by the hemocytes, appearing as a complex family of cells specialized in immunity . These cDespite the important role played by hemocytes in the invertebrates’ immune response, these cells are still very poorly characterized. Different types of invertebrate hemocytes have been described in the literature , mainly Schistosoma mansoni, the agent responsible for human intestinal Schistosomiasis has also benefits from this technology. The scRNA seq approaches on stem cell populations from juvenile Schistosoma allowed to precisely define 9 transcriptomic populations of somatic cells, and the characterization of conserved gene sets involved in the regulation of germline cells (Bombyx mori nucleopolyhedrovirus) on some hemocyte populations , recovered in 1975 and maintained since then under constant laboratory conditions. Snails were maintained at 26°C in glass aquaria and fed with green leaf lettuce ad libitum.We used a ® fluorescent mounting medium  and finally the hemocyte populations and their proportions were counted under light microscopy using a 100X objective (10 snails (size: 8-10 mm) were retained to collect hemocytes by puncturing the hemolymph (30µL per snail) according to a widely used protocol previously established using the defence reflex of hemolymph released from the cephalo-pedal sinus when the snail shrinks in its shell . HemolymPooled hemolymph (500µl) from 50 snails was recovered into 2ml tubes and mixed with 1.5ml of modified anticoagulant solution  . Sample http://10xgenomics.com).Single cell processing procedure is defined by the MGX platform (Montpellier GenomiX). Single cell suspension was obtained by a Chromium Single-Cell Controller (10X Genomics). Library preparation was performed with Single Cell 3’ Reagent kits V3.1 (10X Genomics) using 10x Next GEM Technology barcode and validated by DNA quantification with Fragment Analyzer (kit High Sensitivity NGS) and qPCR (ROCHE LightCycler 480). Libraries were sequenced with an illumina NovaSeq 6000 (Illumina) and SBS (Sequence By Synthesis) techniques using NovaSeq Reagent kits (100 cycles). Output results and matrix generation were processed with 10X Genomics Cell ranger v3.1.0 software   .Biomphalaria glabrata reference genome V1.6.Filtered matrix sorted by Cell Ranger (10X Genomics Cell Ranger 3.1.0) was used and analyzed on R (Version 4.1.0 (2021–05–18)) using Seurat package [Version 4.0.4 ]. HemocyThe cell lineage analysis was performed with bioinformatic tools Monocle3  and slinBiomphalaria glabrata snail (available at vector base: https://vectorbase.org/vectorbase/app). Qualitative differences between samples were considered as potential markers of sorted hemocyte populations and used to infer scRNAseq clusters to a potential morphological population of hemocytes.Pooled hemolymph of 300 BgBRE snails was used for cytometry cell sorting. Briefly, hemolymph was extracted from the snails as previously described and immediately used for flow cytometry and cell sorting, 25 μL of hemolymph was recovered from each snail for a total of 3.5 ml. Cell sorting was performed, using a FACS Canto from BD Biosciences , according to the FSC and SSC parameters to discriminate each hemocyte population according to their size and granularity. With the help of FACSDIVA software, four gates were defined based on different SSC and FSC parameters (low SSC/FSC to high SSC/FSC). Hits detected in these gates were sorted, recovered on microscopic slides and observed under a light microscope to confirm the enrichment of different morphological hemocyte populations associated with the different settings of cell sorting gates. Finally, sorted hemocyte samples were recovered for label-free proteomic analysis. Recovered hemocytes were lysed in hypotonic buffer, protein extracts were quantified (2D-Quant kit protein quantification) and 200 µg of proteins were solubilized in laemmli buffer 4x , boiled at 95-100°C for 5 min, frozen at -80°C and send for label free sequencing to EDyP service facilities . Protein preparation and mass spectrometry-based proteomic analyses were conducted as described in , 44. ProBiomphalaria glabrata. To clarify this purpose, we decided to establish an “hemocytogram” of circulating B. glabrata hemocytes after plating. Three distinct hemocyte populations were identified: hyalinocytes, blast-like cells and granulocytes . We obtain 2624 estimated cells with a median gene per cell corresponding to 1885 genes among the 19 820 total genes detected and a sequencing saturation at 66%. To retain high quality single cell RNA-seq data, we removed low-quality cells with gene number less than 750 and mitochondrial genes less than 5%. Seurat R package (Version 4.0.4) was used for data processing. Following such quality control, 2193 high-quality cells were used for further analysis. For each cell, an average number of 2470 genes expressed were obtained and 7 transcriptomic clusters were identified , the sequencing data were aligned to the Most of the hemocytes gathered within a main cluster (cluster 0) which represented 73.8% of the total cells. The other clusters (cluster 1 to 6) represented from 7.7% (cluster 1) to 1.9% of the cells respectively  for the selected genes compared to all remaining cells  as we can find in cluster 3 but with lower differential expression , 51. OthCluster 1 represents 7.7% of the total cells  as well as genes having immune-related annotations like cathepsin and apolipophorins, an immune receptor role like toll-like receptor or macrophage mannose receptor or even functions annotated to be involved in response to virus  prevent from defining relationships between cells in the other clusters. For this reason, we removed clusters 1 and 6 and re-run the cell lineage clustering analyses  studied, from each of the two morphological hemocyte populations. We then averaged the expression of all genes, by cell type, that we are able to detect in our scRNAseq analysis to determine which transcriptomic cluster would be very likely to express these immune gene pools , suspected to be expressed by some granulocyte subtypes  and flow cytochemistry approaches, or by optical microscopy and cell morphology description  of the hemocytes before analysing them with a global label-free proteomic approach. Nonetheless, as previously shown , B. glabHowever, we decided to remove clusters 1 and 6 from the analyses because the genes expressed in these two clusters are very different from the other clusters. It was then impossible to obtain a cell lineage between these two clusters and the rest of the clusters grouped around cluster 0. Either the cells that compose clusters 1 and 6 are from different progenitors or they are from different cell lines that we were not able to capture by this drop-seq approach. To solve this problem one possibility would be to increase the number of hemocytes captured in order to detect the rare hemocytes in our study model that could link all the transcriptomic clusters in the analysis. With this increase in the number of hemocytes captured, it would also be important to increase the depth and sensitivity of sequencing in order to be able to detect more genes in all cells like the use of well-based scRNAseq methods including smartseq2-3 , 59. ForB. glabrata snails or more largely in invertebrates? This question will deserve further investigations.Cluster 0 regroups the majority of the cells in our analysis and preferentially expresses a multitude of PRRs and genes involved in energy production. The cells of this cluster may be probably be considered as latent, sentinel cells, harboring a large diversity of receptors waiting to be activated by a contact with a stimulus . The cells in this cluster also express all the marker genes of the three hemocyte morphotypes defined on the basis of data from proteomics or the literature. It would therefore seem that this cluster is possibly composed of several different hemocyte morphotypes or only hyalinocytes cells due to the large number of genes expressed which would suggest large cells, waiting for a biotic or abiotic signal to engage in a specific differentiation pathway. One possible scenario would be that some of these cells, from cluster 0, would enter into division (which could represent cluster 4) and then produce active cells (represented here by the cells of cluster 3 linked in the pseudo-time to cells of cluster 4). This differentiation from cluster 0 to cluster 4 and cluster 3, may also be supported by the analysis of the MIF gene  showing B. glabrata snails. This is particularly true for bacteria  or virus (cluster 6). These clusters could represent hemocytes that can be involved in the control of microbiota communities. Indeed, it is yet hypothesized that the immune system is necessary for the control and maintenance of the microbial communities associated with the holobionts in bacteria , 63 and bacteria  in whichAll the results in this study still prevent us from assigning each transcriptomic cluster to a particular hemocyte morphotype with certainty. However, this scRNAseq analysis provides the basis for further descriptions of the function of each hemocyte type. The marker genes defined in this analysis allow us to draw an identity map of each of the transcriptomic clusters, and will be a terrific tool that can be used in a near future for the morphological validation of each of these cell groups. For example, a gene coding for a transmembrane protein (transmembrane 97-like) is highly expressed in cluster 1 and could be of great interest to determine if this cluster corresponds to one or more morphological cell types. But how can we physically separate our hemocytes? How to define more precisely the cells of cluster 0? The next challenges concerning the description of these hemocytes will consist in developing and combining more cutting-edge technology approaches of physical separation of hemocytes. We have summarized in in-situ hybridisation, siRNA or CRISPR-Cas9. Unfortunately, those approaches could still not be relevant to obtain membrane markers that could be used for FACS purposes. Indeed, surface markers often have low levels of RNA abundance and can be under the method sensitivity threshold. For this purpose, we propose to enrich hemocyte membrane proteins for MS-MS analysis to identify proteins harbored on hemocyte surfaces. Those proteins can then be used as targets for antibody-based isolation  or immunohistochemistry. As soon as several sub-populations of hemocyte can be isolated in flow cytometry gates, a novel era may start for the Biomphalaria glabrata community working on immunity. Obviously, it would pave the way for basic flow-cytometry immunology where cell populations are monitored following an immune stress, alarmin injection or parasite infection. The improvement of FACS techniques, coupled with approaches of more sensitive scRNAseq technologies such as smart-seq methods would increase the quality of transcriptome obtained for each population and be useful to characterize each of the morphological populations and their behaviour to certain pathogens or parasites. Moreover, those sorted sub-populations could then be used for in-vitro endocytosis or opsonin assays where we may observe the ability of the different hemocytes to interact, induce phagocytosis or actively endocyte differentially treated beads, parasites or pathogens.These approaches can be split in two strategies, using hemocytes and complex tissues as a bulk or using single isolated cells. More in-drop isolated scRNAseq sequencing would bring new information about response to infection or injury in hemocytes. More sequencing would bring more accurate marker genes that could be used for targeted approaches such as S. mansoni responsible for intestinal schistosomiasis in humans. Much evidence leads to hemocyte while suggesting a support for innate immune memory. Here, we bring new evidence proving the plasticity over the diversity of hemocytes population in Biomphalaria glabrata and we bring new guidelines to transform hemocyte immunology research in invertebrates.Finally, determining the links between the different morphological populations and their functions is an essential step in understanding snail immune mechanisms, particularly in the context of immune response and innate immune memory acquisition towards the parasite The datasets presented in this study can be found in online repositories. The data presented in the study are deposited in the Sequence Read Archive (SRA) repository, accession number PRJNA844512.Investigation and experimental procedures: RP, MP, AP, RG and BG. Bioinformatic analysis: RP, SP, CC. Writing—Original Draft Preparation: RP, SP and BG. Writing—Review & Editing: RP, SP, EV, DD, RG, and BG. Project. Administration: RG and BG. Funding Acquisition: DD and BG. All authors contributed to the article and approved the submitted version.This study is set within the framework of the “Laboratoires d’Excellences (LABEX)” TULIP (ANR‐10‐LABX‐41). DD and BG were supported by the ANR AEROSNAIL (ANR-19-CE11-0016-01) from the French National Research Agency (ANR). BG was supported by the BQR from the University of Perpignan. All authors contributed to the article and approved the submitted version.Thanks to cytometry facilities of the MRI platform (Montpellier ressource imagerie) as well as the EDyP service platform for their help in the development and implementation of cell sorting and label-free proteomics. Thanks to the technical staff of the IHPE laboratory, Jean-Francois Allienne for his expertise in molecular biology, Anne Rognon and Damien Pouzol for the maintenance of mollusc strains and their expertise on animal experimentation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Interestingly, sensor performance improved upon transition from N2 to air, with the inverse being true for most NO2 sensors. UV irradiation both improved sensor dynamics and stabilised the sensor electrical baseline, allowing sensors based on SilPEG to fulfil the requirements of sensing solutions used in industry (below 10% baseline drift after sensors reach saturation) and making them promising candidates for further development and applications. Based on conducted multi-variate experiments, an initial mechanism underlying the interplay of exposure to oxygen (present in air) and UV irradiation was postulated.Nitrogen dioxide (NO Nitrogen dioxide is an unfortunately frequent contaminant of air, both at industrial sites and in residential zones. NO2 has been found to cause adverse effects to human health -co-[poly(MHS)-graft-PEG]) (SilPEG) and (Poly(DMS)-co-[poly(MHS)-graft-2-vinyl-poly(3-hexylthiophene)]-co-[poly(MHS)-graft-dodec-1-en] (DodecSIL). The scheme of the synthesis of the obtained rrP3HT graft copolymers is presented in The synthesis of the vinyl-terminated regioregular poly(3-hexylthiophene) (vin-rrP3HT) via the Grignard metathesis method was conducted based on the procedure described in the literature . All of lication  The synt1H NMR spectroscopy). The characteristic signals are summarised in the The chemical structure of the obtained materials was confirmed by Fourier transform infrared–attenuated total reflectance mode spectroscopy (FT IR ATR) and proton nuclear magnetic resonance spectroscopy  spectrometer with a resonance frequency of 300 MHz using tetramethylsilane (TMS) as an internal standard. FT IR spectroscopy was carried out on a Perkin–Elmer Spectrum Two  spectrometer with a UATR (single-reflection diamond) module. The average molecular weights were determined for tetrahydrofuran solutions of sample concentration 1 mg/cm3 using a size-exclusion chromatography (SEC) 1100 Agilent  1260 Infinity isocratic chromatograph with a differential refractometric MDS RI Detector (USA), calibrated by linear polystyrene standards .Ultraviolet–visible–near-infrared (UV-Vis-NIR) spectra were registered for polymer films deposited via spin-coating with a typical spectrum acquisition time of 25 ms and with 40 individual spectra being averaged to improve the signal-to-noise ratio. UV-Vis-NIR spectra were recorded using a diode array detector spectrometer set supplied by Ocean Optics that consisted of two measurement units, QE65000 and NIRQuest 512  and a DH-2000-BAL balanced deuterium and halogen light source.The potential was applied using step chronoamperometry, with the sample initially being conditioned by the potential used in the previous step  for 5 s, following which the potential used in the current step was applied. Conditioning with this second potential was performed for approximately 100 s in order to ensure that all processes taking place in the sample were at equilibrium. Following that, the spectrum for the given applied potential was recorded. A Metrohm Autolab PGSTAT302N potentiostat was used for the purpose of applying the potential staircase to the experimental cell.2 dielectric layer. A photolithography process and physical vapour deposition were used to create electrodes on the surface of the transducer. Firstly, 20 nm of chromium used as an adhesive layer was evaporated, which was covered with a 100 nm layer of gold. The width of the electrode was 3 μm, the distance between the fingers was 10 μm, and the length of the channel was 1500 μm. Thin-film conductive polymer layers were created with the drop coating method. A total of 2.5 mg of conductive polymer material was dissolved in 1 mL of chlorobenzene  using 30 min of ultrasonic stirring. The resulting solution was applied to the IDT with the drop coating method using a micropipette , applying 6 μL of the solution to the substrate for the given type of conductive polymer. Elaborated structures were left to dry completely under a laminar chamber for 12 h on the heater plate and purged with pure nitrogen gas to remove non-adhering materials. Elaborated structures were placed on an Al2O3 electric heater and connected to chip feedthroughs via ultrasonic bonding, using a 25 μm gold wire. The bonding process was realized using a 53XX-BDA, F& K DELVOTEC wire bonder. The morphology and roughness of the obtained thin films were investigated using atomic force microscopy (AFM) NTEGRA Prima . Topographic imaging was performed in semicontact mode using an HA_NC ETALON cantilever from NT-MDT . Simultaneously with topographic images, the AFM phase shift was recorded.In order to carry out gas measurements, conductive polymer thin film sensor structures on interdigital transducers (IDT) were fabricated. The IDTs were created on a 2O3 heater. Measurements were carried out at room temperature, both in dark conditions and under constant UV irradiation -type chip to conduct gas tests in different gaseous atmospheres. Resistance measurements of the sensor’s active polymer layers were conducted using a multichannel switch unit 34970A , whose measurement range was set to 100 MΩ, in which a 500 nA current source is used. Temperature (23.5 °C) was measured by proportional–integral–derivative (PID) controller SR94  using a Pt100 sensor attached to an AlThe acquisition of measurement data and the preparation of the gas mixtures were performed by a gas mixing server, utilising mass flow controllers  controlled by LabVIEW environment, which allows for precise preparation of gaseous atmospheres. During the measurement process, the relative humidity (RH) of the gas mixture was measured by a RH meter. The RH was maintained at a level of 3.5% ± 1% (at 23.5 °C).2 (from gas cylinder with standard mixture of NO2 1000 ppb in N2) for each nitrogen dioxide measurement cycle. The sensors were also exposed to other gases , in order to determine cross-sensitivity of the sensors. In this case, 10% CO2 and 10 ppm NH3 an RH 50% @ RT were ran in the measurement cycle, respectively. Regardless of the utilised carrier gas or gas mixture, a constant volumetric gas flow of 400 mL/min was maintained throughout all of the experiments. The short-term baseline drift values were calculated as the values of the response after 30 min of sensor recovery (average from last 20 s); thus, they show the difference between the response value after a recovery cycle and the initial baseline .The gas detection measurements were divided according to the type of the used carrier gas (nitrogen and synthetic air) and also according to the conditions of stimulation of the sensor structures with UV radiation . One measurement cycle consisted of alternation between exposing the sensor structures to the mixture of a carrier gas and the tested gas (the mixture was prepared so as to have the specified concentration of the tested gas) for 30 min and purging the sensor with the carrier gas for 30 min. The following doses of nitrogen dioxide gas were used: 50, 100, 200, 300, 400, 500, and 1000 ppb of NOgR is the resistance measured in the target gas .aR is the resistance measured in carrier gas .The sensor response for reducing and neutral gases was calculated by using the Formula :(1)RespoaR is the resistance measured resistance in carrier gas .gR is the resistance measured in target gas .For oxidising gases, the sensor response (R) was calculated using Formula :(2)Resporesp90%) was calculated as the time taken to reach 90% of the sensor’s maximum response value. The recovery time (trec10%) was calculated as the time from start of clean carrier gas flow to reach a level of 10% of the maximum response value.The response time  for 2400 s. Tests were conducted in dark conditions and under UV irradiation (2), the sensing structure still does not achieve a stable response value below 10% of the maximum response value. Under activation by UV, a stable signal was achieved after less than 20 min. The response of the sensing structure after 400 s did not stabilise in either dark conditions or under UV irradiation. Similar results were obtained for the DodecSIL-sensing structure. Based on these results, we selected 30 min to be the duration for exposure and recovery cycles, as this period is sufficient to present the sensing characteristics of the investigated materials.The significant recovery time for polythiophene-based sensing structures exposed to NOadiation . The obt2 carrier gas). In this case, the polymer films had very high base resistances (exceeding 100 MΩ) and were out of range for the standard measurement unit used. The resistance in N2 at dark conditions was examined using Keithley 4200-SCS parameter analyser, and its value was around 250 MΩ for SilPEG. NO2, being a strongly oxidising gas, doped the polymer, resulting in its electrical resistance value decreasing to less than 100 MΩ after exposure to 100 ppb for 30 min. The response of sensors using the two copolymers to NO2 concentrations in the range of 200–1000 ppb is shown in  and are much higher than DodecSILs ones. For both polymers, responses to NO2 in air (dark) are higher than its responses in N2. This is due to the additive effect of oxidation by NO2 and O2. When UV activation is applied (In 2 concentration) are presented. In nitrogen carrier gas . In such a situation, we can only consider the regeneration process in the case of SilPEG in air atmosphere with UV; therefore, the analysis of the response time and the recovery time was carried out only for SilPEG in air with UV, which showed the highest response and stability  and neutral (CO2) gases (2 (over 144% to 1 ppm) relative to that of NH3 (over 123% to 10 ppm). SilPEG for all tested gases showed a higher sensitivity than DodecSIL.Selectivity was also determined for the interaction of sensors with other common gas analytes, including reducing (NH2) gases b. The inThe impact of illuminating the sensor structure of SilPEG with UV light was measured using a Keithley 4200A-SCS parametric analyser with a 1 V DC bias. The measurement cycle was designed to provide more information on the interplay of effects caused by exposure to air and irradiation with UV.2 atmosphere. This irradiation leads to the photogeneration of charge carriers, which is observed as an increasing current flowing through the sensing structure. The current increase is initially fairly rapid, but quickly slows down as the concentration of photogenerated charge carriers becomes sufficient for their recombination to become significant. Once the N2 atmosphere is replaced with air, another similarly shaped current increase is observed, but with a much greater magnitude than for the N2 atmosphere. Once air is replaced with N2,this increased current quickly decays and gradually stabilises at an intermediate level.In the first experiment a, the se2, as seen by the much higher currents flowing through it in air than in N2. Once UV irradiation was started, the current flowing through the sensor increased almost identically to the case of the first experiment. This current quickly diminished when UV irradiation was ceased.The second experiment was conducted entirely in air and was set to examine the influence of UV irradiation on the electrical conductivity of the sensor b. It sho2 in synthetic air carrier gas over time. The response of the “as deposited” sensing structure after 1 h of illumination was 260% but decayed to 145% and 18.5% after one day and two weeks, respectively , which is the key electroactive component of both copolymers. Upon oxidation, absorption signals corresponding to doped polymer segments begin evolving, centred at approximately 750–770 nm, as well as at lower energies, with only an onset of the signal being observed.2 sensor configurations, but may have slightly lower responses than SilPEG, likely due to the fact that it undergoes dedoping more rapidly.Once the applied potentials are gradually decreased, the doped polymer signals begin decaying and the ground state (undoped) polymer absorption begins recovering. This recovery, however, is only partial, indicative of limited doping state reversibility, with the achievement of high doping levels possibly being tied to degradation of the conjugated system as no doped polymer signals remain after −0.5 V is applied as the final conditioning potential. This doping/dedoping irreversibility is slightly less pronounced for DodecSil  than for SilPEG . Because the interactions of the copolymers with nitrogen dioxide are likely to cause repeated doping and dedoping, DodecSil is likely to exhibit longer lifetimes in NOThe above may also serve to explain the differing effects of UV irradiation on sensor dynamics in nitrogen and in air, as oxygen can interact with spin-bearing  charge carriers present in doped conjugated polymers, whereas nitrogen does not form such interactions. Upon irradiation, the oxygen–charge carrier interactions may be disturbed, possibly leading to a decomposition of the charge carrier and a return of the polymer segment to the ground undoped state.2 is higher than that of DodecSIL, regardless of N2/air atmosphere or the presence/absence of UV irradiation.In general, the response of SilPEG to NOa) of the sensors. On the one hand, this lowered base resistance helps control baseline drift . The observed activation of the receptor layers in air upon exposure to UV irradiation is tied to the photogeneration of charge carriers on the polymer chain. This photogeneration takes place via the absorption of a quantum of energy (hν) to induce charge separation on neighbouring repeat units of the polymer (Th) and is a well-known phenomenon [Interestingly, the effects of both UV and air exposure are not entirely straightforward, as in the case of these poly-based receptor layers, an interplay of the two factors is observed , even prenomenon . If the It should be noted that photogeneration of charge carriers is accompanied by an opposing process: recombination of charge carriers. At steady conditions, the two processes eventually achieve equilibrium, resulting in the observed, gradually decelerating increase in current flowing through the sensing structures  when thePolythiophenes are p-type conductors, that is, their conductivity is realised primarily via the movement of positive charges which are, in this case, expressed as radical cations  and the recombination of positive and negative charge carriers (charge-carrier decay). The increase in current observed upon exposing the sensing structures to air results from a significantly higher charge-carrier population, indicating that a new photogeneration/recombination equilibrium state being established in the polymer. Because during the aforementioned experiments the irradiation intensity was kept constant, the rate of charge-carrier recombination must have been decreased once the sensing structures were exposed to air.2, resulting in the formation of a radical cation on the polymer chain. We hypothesise that in our case, oxygen present in air interacts with the negatively charged repeat units in our polymers as per the following reaction , but in the case of negative charge carriers, will result in a charge-transfer complex −·. Consequently, such a “bonding” of negatively charged photogenerated charge carriers would hinder recombination while maintaining the presence and relatively high mobility of positively charged charge carriers In the case of photogenerated charge carriers, not only will such interactions have a greater driving force  concentration. However, the gradual deactivation of the receptor material in these conditions necessitates either employing an intermittent operating regime of prospective sensors or preventing this deactivation, at least partially, e.g., by introducing additives capable of absorbing excess UV radiation into the polymer receptor material matrix. Metal oxides, such as zinc oxide, are one prospective type of additives that could significantly improve the resistance of the receptor material to UV irradiation and perhaps even improve the performance of the sensing structures. Such functionalization of proposed polymers could also solve the problems with the long-term stability and improve the selectivity and response dynamic so that applications may be found in the automotive and energy industries, as well as in air quality monitoring.To summarise, not only did UV activation significantly improve the dynamics of operation of the sensing structures in experiments using air as a carrier gas, but it also helped minimise the short-term baseline drift for the sensing structures, particularly for ones based on SilPEG. UV activation for SilPEG allowed for a short-term baseline drift below 10% after the sensing structures reach saturation at a given NO2/air carrier gas exchange allowed us to formulate an initial postulate about the processes taking place within those receptor layers. The proposed mechanism explains the obtained experimental results to a better degree and is more in line with the properties of conjugated polymers than the most commonly postulated mechanism based on the features of inorganic semiconductors that involves photo-stimulated desorption of oxygen from the sensing structure [The use of experiments alternating UV activation and Ntructure .


The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here, we developed a human 2D patient derived organoid screening platform to study tissue patterning and kinase pathway dynamics in single cells. Using this system, we discovered that waves of ERK signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If ERK is activated acutely across all cells instead of in wave-like patterns, then tissue patterning and stem cells are lost. Conversely, if ERK activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated ERK signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how ERK can antagonize stem cells while supporting cell replacement and the function of the gut. The colonic epithelium is a highly organized and rapidly renewing tissue containing spatially separated compartments called crypts. Roughly 10 million evenly spaced crypts inhabit the colon and are renewed every 3–5 days . Each coDrosophila embryos, ERK signaling promotes differentiation if the signal is prolonged or total strength is increased  cells found that cells at the wound barrier initiate ERK signaling cascades which propagate in wave-like patterns across the cell monolayer away from the site of damage. These ERK waves drive collective movement in the direction of the wound to efficiently seal barriers . ERK wavncreased . A recenncreased . In summncreased . Yet, itLGR5 and MYC. Alternatively, if ERK is inhibited globally, nodes increase in size and express high levels of LGR5 and MYC. If Wnt3a is removed from culture media, nodes also disappear and ERK activity is increased. These data show that Wnt and ERK mutually oppose one another to maintain gut homeostasis and Wnt-suppressing ERK waves originating from apoptotic cells help to shape the architecture of the human colonic epithelium.Herein, using self-organizing primary human colon organoid monolayers, we show that epithelial cells can reorganize from single cells into a complex monolayer resembling the tissue of origin. Regularly spaced crypt-like structures (hereafter referred to as ‘nodes’) form 3–4 days after cell dissociation and seeding. These nodes are maintained for weeks in spatially distinct compartments. In non-node compartments, cell extrusion and apoptosis are increased. As a cell is extruded and undergoes apoptosis, an ERK wave is initiated that propagates radially from the dying cell. Locally, cells in the path of the wave are prompted to migrate toward the dying cell, presumably to maintain epithelial integrity as reported recently . The spaintestinal Line, Arizona 1) formed regularly spaced nodes, quickly reached confluence within 72 hr, and could maintain node structures for 45 days or more. When GiLA1 single cells were seeded onto an ultra-thin layer of Matrigel, organoids formed monolayers by 3 days and began to spontaneously form regularly spaced compact compartments reminiscent of colonic crypts within 3–4 days  to form 5–10 cell clusters. After this, cells became hyperproliferative from 48 to 72 hr after seeding to form a complete monolayer. Finally, morphologically distinct cellular compartments were formed by 96 hr . These 2LGR5 and MYC expressed at higher levels in nodes compared to non-nodes  to acutely activate ERK signaling across the monolayer. Following treatment with 100 nM PMA, we observed immediate hyperactivated ERK in all cells . Monolayers showed a remarkable loss of patterning after PMA treatment as evidenced by almost complete loss of cell clusters by 24 hr post-treatment  between neighboring proliferative compartments and the average distance (~447 μm) covered by the apoptosis-induced ERK wave . This reG12V system (G12V expression (G12V induction resulted in a ~twofold reduction in nodes after 7 days (G12V was induced after organoids formed monolayers but before node formation (on day 2 after seeding), KRASG12V expression led to a lack of node development in organoid monolayers (PMA is a known activator of protein kinase C (PKC) and could change cellular pathways driven by PI3K, MMPs, FAK, and calcium . To testV system . Within V system . OncogenV system . In linepression . Patternr 7 days  and compr 7 days . If KRASnolayers . These dWnt is a well-known promoter of stem cell renewal within the gut and may be responsible for the clustering of the proliferative nodes as we previously observed in murine small intestine monolayers . We askeThe human colon requires ongoing regeneration and renewal over a lifespan; this need relies on finely tuned communication between differentiated and stem cells. We show here that one way the colonic epithelium maintains both complexity and plasticity is through long-distance signaling driven by cell turnover. As cells differentiate and are eliminated from the epithelium, dying cells initiate an ERK wave that triggers local cell tropism towards the death event and stimulates differentiation of replacement cells. This process is an efficient way for the colon to maintain the correct proportions of stem and differentiated cells in distinct spatial compartments.We have shown that primary human colonic tissue can be expanded and transformed into monolayers that phenocopy key characteristics observed in the tissue of origin. Although the immune and stromal compartments are not included in this culture method, different compartments of stem and differentiated cells can be established and form unique physical properties depending on the source of the tissue  and S1. LGR5 and MYC , and myosin light chain kinase (MLCK) are essential for apoptosis-driven membrane blebbing  and extr+ stem cells , or core needle biopsies were collected from consented patients by the  facility located in the Arizona Health Science Center. Primary colonic organoid cell lines were anonymized by the Tissue Acquisition and Cellular/Molecular Analysis Shared Resource (TACMASR) at the University of Arizona Cancer Center. TACMASR is an on-campus biorepository to procure, store and retrieve biospecimens in a form that is deidentified and protects the privacy of the donors and confidentiality of the data collected. The individuals from whom the cells originated were resection patients at Banner University Medical Center. All research participants in this proposal receive the cells with de-identified and anonymous labels that cannot trace back to the individual or their families from which they came. Thus, no one involved in this study can access the subject’s identities. Therefore, the study is exempt from being considered human subject research. Biopsy tissues were transported in 50 ml conical tubes containing collection media  at the BioDROids core facility, located in the University of Arizona Cancer Center. Frozen tissues were prepared into organoids by thawing tissue pieces, mincing, and incubation with 1 mg/ml collagenase type 3 in phosphate-buffered saline (PBS) on a shaker for 10–25 min at room temperature. Cells were then removed from collagenase, washed with PBS, embedded into 100% Matrigel, and cultured in seeding media for 7 days. After organoids became established, low passage aliquots were cryopreserved in freezing media or infected with lentiviral constructs.https://pdmr.cancer.gov/). The set were selected on the basis of microsatellite stable and APC mutation-negative status (seven organoids) and a set of microsatellite stable and APC mutation-positive organoids were matched to the former set on the basis of age, gender, location, and stage. Two organoids from microsatellite unstable tumors were also selected, one APC mutation-negative and the other APC mutation-positive. In the present study, APC mutation-negative organoids 555926–031 R-V1-organoid and 624824–186 R-V1-organoid, APC mutation-positive organoids 276233–004 R-V1-organoid, 451658–271 R-V1-organoid and 722911–139 R-V1-organoid, and the microsatellite unstable organoid 616215–338 R-V2-organoid were expanded in Matrigel and organoid media according to the PDMR’s published protocols.A set of 16 colorectal cancer organoid models were purchased from the National Cancer Institute Patient-Derived Models Repository  supplemented with 2 mM GlutaMax, 10 mM HEPES, N-2 media supplement (Invitrogen 17502048), B-27 Supplement Minus Vitamin A (Invitrogen 12587010), 1 mM N-Acetyl-L-cysteine (Sigma A9165), 2500 units/mL Penicillin and 2.5 mg/mL streptomycin (0.05 mg/mL), 50% L-WRN Conditioned Media , 100 ng/Complete LWRN media supplemented fresh with 2.5 µM CHIR99021  and 10 µM Y27632 .6 HEK 293T cells were seeded in a 10 cm plate and transfected the following day using 500 μL of opti-MEM, 30 μL Gene Juice (Sigma #70967), 5 ug lentiviral construct, 3.25 µg psPAX2 (addgene #12260), and 1.75 µg pMD2.G (addgene #12259). After 24 hr, fluorescence was assessed, and media was collected for 3 days followed by centrifugation and filtration using 0.45 µM syringe filter. Lentiviral media was then kept at 4°C before 100 × concentration using LentiX concentrator (Takara #631232) and resuspension of virus in organoid seeding media; viral media was then stored at –80°C for up to 3 months. For infection of organoids, 1×105 primary epithelial cells were harvested from 3D domes, washed, and trypsinzed as described previously. Cells were then counted using a hemocytometer and 40 K organoid cells were plated in suspension onto a 48 well plate and diluted 1:1 in vial media containing 8 mg/ml polybrene. Next, organoids were placed at 37°C for 1 hr the before centrifugation at 600 g for 1 hr at 32°C. Organoids were then harvested, washed, and embedded into 90% Matrigel and cultured in seeding media for 24 hr. Cells were then grown in complete LWRN for 3 days before fluorescence was assessed and >50% infection was achieved. Media was aspirated and organoids were resuspended in 500 ul trypsin. After dissociation, cells were mixed 5–10 times using a p200 pipette tip and passed through a 100 μm filter to ensure isolation of single cells.3×10SCREENSTAR 384-well black plates (Grenier #781866) were coated with ice cold Matrigel (CB40230C) diluted 1:40 in Serum-free Advanced-DMEM F12 media (SFM) for 1 hr. Monolayers were prepared using our previously described protocol . Briefly>the mean ERK-KTR nuclear signal. The density-based spatial clustering of applications with noise algorithm  was performed on the centroids of ERK active cells to identify spatial clusters of cells with active ERK. A boundary was drawn around the spatial clusters using the boundary algorithm in MATLAB. The ERK positive regions of each frame were summed up to generate the heat map in MATLAB.384-well plates were imaged with fluorescent microscopy on a Nikon Eclipse TI2 automated microscope. For quantification of wave dynamics, organoids were imaged every 10–30 min. Tracking and segmentation of single cells in time lapse images in i at timepoint t  was a gift from Roel Nusse, addgene plasmid #24304 .Goat polyclonal anti-Muc2 Alexa Fluor 546 Phalloidin Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye DAPI Rat monoclonal anti-Ki67 Rabbit polyclonal DCAMKL Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Donkey anti-Goat IgG (H+L) Secondary Antibody [DyLight 488] (Pre-adsorbed) NucView Caspase-3 Enzyme (Biotium #10403)After infection with lentiviral reporters, dual reporter organoids were derived. Organoids were dissociated into single cells and sorted for similar levels of expression for the desired reporter (see figure S1C for representation of FACS plots generated using FloJo V10). Cell sorting was performed using the BD FACS Aria III flow cytometer.Experiments were done using previously validated and custom RNA probes for target mRNA of interest using molecular instruments HCR RNA FISH protocol for mammalian cells . Briefly This work employs timelapse microscopy to study behavior of human colonic organoids in monolayers as the organoids initially self-organize. The authors then follow maintenance of organization into densely clustered nodes that have increased cells in cell cycle and the remaining more sparsely populated regions with fewer cycling cells. This study builds on a literature demonstrating roles for signaling pathways like ERK in epithelial patterning that have been examined in the cell competition field and, more specifically, in mouse intestinal organoids. This manuscript should be relevant to a broad readership interested in how epithelial organoids can self-organize and the role of specific signaling pathways in the process. In addition, the technical aspects of the work with live human monolayer cultures observed over timelapse with extensive quantification of cell behavior represent a useful advance in the field. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Live-Cell Imaging in Human Colonic Monolayers Reveals Erk Waves Limit the Stem Cell Compartment to Maintain Epithelial Homeostasis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.eLife will include a response to the reviewer critiques and further examination of additional signaling pathways, in particular the Wnt pathway.Essential Revisions to make the paper suitable for Reviewer #1 (Recommendations for the authors):– The methods say "Cytoplasmic regions were defined by a donut-shaped ring _two pixels wide.." Is this supposed to read "at least 2 pixels wide"? Otherwise, it isn't clear how this is done, as cytoplasms would be greater than 2 pixels.– Perhaps this should be in the public comments, but it seems to me in some of the sequences (most notably Video 3a) that the initial event (prior to caspase 3 positivity) is an expansion of the soon-to-be-apoptotic cell with actual outward movement of surrounding cells and then (pertinent to point 1 in the public comment) the expected actomyosin contraction that appears to bring the neighboring cells in tight as much of the apoptosing cell body rises up above the monolayer (as can be seen in brightfield). Yet, there is essentially no blue in the movement heatmap in Figure 3D. How can cells always move towards an apoptotic cell and never away (even randomly)?– The PIV map in Figure S5 might be moved into main figures, as without some aid, readers may struggle to understand how cell motility is turned into the graphic representation. Indeed, Figure 3 is confusing until the supplemental videos are watched and all the supplemental data viewed. It would be great, if some sort of cartoon or guide to how movement and wave work could be provided here, or at least, say an explanation of what a node and what movements are measured can be shown in Fig, 3C. I spent a long time looking at individual cell clusters to see their relative positions over time to begin to understand the data.– What is Figure 4H relative to? Is this in inhibitor experiments or just control?– It is unclear what Figure 5F represents? What is "Sum/Cell"? Again, as above, differences in PMA-treated cultures may be due to cell death.– There seemed to be scale bars in some of the videos, but I didn't see where their magnitude was given.Reviewer #2 (Recommendations for the authors):It's hard to find information on the number of independent biological organoid lines used in this study. It's important that this information be included. A cell shouldn't be considered a replicate.Figure S4, can the concentrations be included? Was a dose-response curve used to determine the concentrations?The link between apoptosis and tissue patterning proposed in the second paragraph of the "Erk waves instruct pattern" isn't supported by data. A disruption of apoptosis would need to be performed in order to make such a claim.In the discussion, the "Organoid Monolayer System" section suggests that there may be a role for Erk signaling in tissue origin memory. This is not well supported by literature and ignores a lot of work on developmental patterning and epigenetic control of tissue identity including experiments by Nicole Le Dourain and other experimental embryologists that go back decades and would argue against a role for kinase signaling as a key to maintain tissue patterning.Reviewer #3 (Recommendations for the authors):1) The authors should provide a better characterization of the different cell types present in their organoid monolayers, such as Goblet cells, enteroendocrine cells, enterocytes, Tuft cells etc.2) Is the intensity and duration of the Erk waves dependent on the amount of apoptosis?3) It would be worth confirming these findings using the authors' tumor line in addition to their GiLA1 line. They could then ask whether the lack of node formation in their tumor line could be reversed, such as via the addition of ERK inhibitors, if those cells have a high Erk activation, as was suggested in the previous literature.4) Since the authors argue that the Erk and Wnt pathway are mutually antagonistic, it would be informative to quantify Wnt activity during the Erk waves. This quantification could be performed with regard to space and time relative to the Erk waves. This experiment, while not essential for the current findings to have merit, would significantly help our understanding how those two pathways interact.5) The -Wnt3a phenotype should be confirmed by using a Wnt3a inhibitor.6) Figure 4B: please add a control image. Reviewer #1 (Recommendations for the authors):– The methods say "Cytoplasmic regions were defined by a donut-shaped ring _two pixels wide.." Is this supposed to read "at least 2 pixels wide"? Otherwise, it isn't clear how this is done, as cytoplasms would be greater than 2 pixels.Sorry for the confusion. It is true the cytoplasm is greater than two pixels. Yet, automated cytoplasmic segmentation often incorporates errors, especially in dense, confluent cultures. We found that a two pixel annulus adjacent to the nucleus was the most accurate cytoplasmic measurement. Since the reporter is diffuse in the nucleus the mean of this region is a good proxy for the concentration of the reporter in the cytoplasm. This approach is standard for KTR reporters and is used by other groups, in particular the developer of the ERK-KTR. For further details the following references describe this method in more detail:Kudo, T., Jeknić, S., Macklin, D. et al. Live-cell measurements of kinase activity in single cells using translocation reporters. Nat Protoc 13, 155–169 (2018). https://doi.org/10.1038/nprot.2017.128Regot S, Hughey JJ, Bajar BT, Carrasco S, Covert MW. High-sensitivity measurements of multiple kinase activities in live single cells. Cell. 2014 Jun 19;157(7):1724-34. doi: 10.1016/j.cell.2014.04.039.– Perhaps this should be in the public comments, but it seems to me in some of the sequences (most notably Video 3a) that the initial event (prior to caspase 3 positivity) is an expansion of the soon-to-be-apoptotic cell with actual outward movement of surrounding cells and then (pertinent to point 1 in the public comment) the expected actomyosin contraction that appears to bring the neighboring cells in tight as much of the apoptosing cell body rises up above the monolayer (as can be seen in brightfield). Yet, there is essentially no blue in the movement heatmap in Figure 3D. How can cells always move towards an apoptotic cell and never away (even randomly)?We agree with the reviewer’s description of the movement following the apoptotic event and have addressed their concerns by describing it more clearly in the Results section, (page 7 top) and adding a description of the different analyses in the methods section with formulas for our calculations (page 4). We apologize for not including this in the original submission.To clarify, during the apoptotic event there is a slight movement away from the apoptotic cell, predominantly in the cells that are very close to the apoptotic cell. This slight movement away from the apoptotic cell is followed by movement towards the apoptotic cell, with the cells closest to the apoptotic cell moving before the cells that are further away. Importantly, since the movement away from the apoptotic cell is much smaller in magnitude than the subsequent movement towards the apoptotic cell it is hard to visualize in the heat maps. Therefore, we used two heat maps to depict cell movement following the apoptotic event.In the middle heat map of Figure 3D, we measured the net movement of each cell toward the location of the apoptotic cell when it died. Since this measurement describes total distance traversed, a cell can be closer to the apoptotic cell in a given frame even if it migrated slightly further away in the current frame as compared to the previous frame. An example calculation would be as follows: if a nucleus was 20 μm away from the apoptotic nucleus at the time of death, and 10 μm away from the apoptotic cell at frame 11, this value would be 10 μm (20 μm – 10 um) for frame 11. If in the next frame, the nucleus was now 15 μm away the value would be 5 μm (20 μm – 15 um), even though it moved slightly further from the apoptotic nucleus. Thus, this calculation measures overall movement towards the apoptotic nucleus and does not capture the frame-to-frame movement.To capture the frame-to-frame movement, we took the values described in the previous paragraph and subtracted frame t-1 from frame t. So, in the example described above, when the cell moved 5 μm away from the location of the apoptotic cell, this value would be -5uM, even if overall the cell has moved closer to the apoptotic cell since the time of the death event. This analysis is depicted in the heat map on the right in Figure 3D. You can see the movement away from the apoptotic cell in the cells closest to the apoptotic cell (top of the heat map). Also, before the apoptotic event the movements of each cell can be both away from the apoptotic cell and towards it, more or less at random as the reviewer suggested would occur.– The PIV map in Figure S5 might be moved into main figures, as without some aid, readers may struggle to understand how cell motility is turned into the graphic representation. Indeed, Figure 3 is confusing until the supplemental videos are watched and all the supplemental data viewed. It would be great, if some sort of cartoon or guide to how movement and wave work could be provided here, or at least, say an explanation of what a node and what movements are measured can be shown in Fig, 3C. I spent a long time looking at individual cell clusters to see their relative positions over time to begin to understand the data.Agreed. We have moved the PIV analysis to the main figures and have added a more detailed description of what is measured in the PIV analysis in the methods section and included a reference.– What is Figure 4H relative to? Is this in inhibitor experiments or just control?We agree this was not the best data representation. Figure 4H was showing the heterogeneity of wave size across all controls, no drug treatment was used. This figure was removed from the main figure and a more detailed analysis was performed and included in figure S5 in order to compare these distances directly to spacing of node regions.– It is unclear what Figure 5F represents? What is "Sum/Cell"? Again, as above, differences in PMA-treated cultures may be due to cell death.Thank you for pointing this out. We have relabeled it LGR5 intensity/Cell. This measurement is made by first segmenting individual cells and quantifying the LGR5 mRNA FISH intensity in the individual cell ROIs. For this reason, effects on cell death would not confound this data. The histogram is a minimum of 5000 single cell measurements showing the distribution of the RNA levels under the different treatments.– There seemed to be scale bars in some of the videos, but I didn't see where their magnitude was given.We thank the reviewer for catching this. The magnitude of the scale bars is 100 microns. This has been added to each of the video legends. Figure 5 was re-made and a 100-micron scale bar was added.Reviewer #2 (Recommendations for the authors):It's hard to find information on the number of independent biological organoid lines used in this study. It's important that this information be included. A cell shouldn't be considered a replicate.Agreed. We used a single patient derived organoid line for the majority of this study, yet we did compare to other patient derived lines that are normal and tumor in origin. We have added this information in the first paragraph of the Results section. We have also added a supplemental figure  comparing five different tumor lines to the Gila1 line to show patterning differences. We added an additional representative image of a normal patient derived monolayer to figure S1. In all experiments, technical replicates (multiple wells) and biological replicates (separate experiments) were performed. In some cases, the data is shown as each data point representing a single cell in order to show the distribution of response across single cells. The details for this can be found in the excel sheet provided with the exact number of replicates for each experiment. We also have added greater detail to the figure legends for number of replicates performed.Figure S4, can the concentrations be included? Was a dose-response curve used to determine the concentrations?A concentration of 1uM was used for each of the drugs. This was to show as was shown previously, that the KTR will shut off in response to well-established small molecule inhibitors. Minimal cell death was observed in the first 24 hours and cells showed clear inhibition of the KTR activity, so dose responses were not performed.The link between apoptosis and tissue patterning proposed in the second paragraph of the "Erk waves instruct pattern" isn't supported by data. A disruption of apoptosis would need to be performed in order to make such a claim.This is an excellent point and indeed it was previously shown that caspase inhibitors do not suppress Erk waves . Though we do believe that apoptotic events will influence patterning more detailed experiments potentially using optogenetic tools are needed to address this question directly. We have toned down the wording of this portion of the discussion to address this concern. Instead, we focus on the role of Erk waves in controlling patterning by replacing “apoptosis” with Erk waves in several places.In the discussion, the "Organoid Monolayer System" section suggests that there may be a role for Erk signaling in tissue origin memory. This is not well supported by literature and ignores a lot of work on developmental patterning and epigenetic control of tissue identity including experiments by Nicole Le Dourain and other experimental embryologists that go back decades and would argue against a role for kinase signaling as a key to maintain tissue patterning.during homeostasis. It would be very interesting to see if epigenetic modifications were maintained after cells stopped producing Lgr5 for example in response to Erk activation and to detail their ability to reprogram back into stem cells, this is an area we are actively exploring. To address this concern, we have toned this section down and highlighted epigenetic modifications as the current major driver of tissue origin memory.We agree. Our intention was not to ignore well-established fate regulation mechanisms such as histone modifications at promoters and enhancers or the beautiful early lineage experiments of LeDourain and colleagues. Our work here simply suggests that Erk waves may help regulate cell plasticity and the architecture of the colon Reviewer #3 (Recommendations for the authors):1) The authors should provide a better characterization of the different cell types present in their organoid monolayers, such as Goblet cells, enteroendocrine cells, enterocytes, Tuft cells etc.Agreed. We have added the percentages of different cell types in the Gila1 monolayers . We quantified stem cells, proliferative cells, colonocytes, tuft cells, and goblet cells. The GiLA1 line showed relatively low levels of goblet cells and slightly higher levels of stem cells, presumably due to its origin from an adenoma.2) Is the intensity and duration of the Erk waves dependent on the amount of apoptosis?Single waves were analyzed, all of which originated from a single death event. We found that if multiple cells died or a tear was formed in the monolayer, waves still occurred. We did not observe enough multiple cell deaths to quantify, presumably due to the anti-apoptotic nature of the Erk waves . We did observe waves colliding with one another, but this did not change the size or duration of the Erk activity in each cell. This could be due to a maximal signal threshold being reached, or a limitation of the KTR reporter’s dynamic range.3) It would be worth confirming these findings using the authors' tumor line in addition to their GiLA1 line. They could then ask whether the lack of node formation in their tumor line could be reversed, such as via the addition of ERK inhibitors, if those cells have a high Erk activation, as was suggested in the previous literature.We agree this would be a fascinating area to study. We addressed this issue by testing the effect of inducible KRAS on the patterning of the monolayers . We attempted to rescue patterning in the KRAS mutant organoids with a Mek inhibitor, but found that the KRAS-induced organoid monolayers became hypersensitive to Mek inhibitor and saw an increase in cell death. We feel to fully address this observation, which is reminiscent of “oncogene addiction” requires a careful secondary study that would be beyond the scope of our work here.4) Since the authors argue that the Erk and Wnt pathway are mutually antagonistic, it would be informative to quantify Wnt activity during the Erk waves. This quantification could be performed with regard to space and time relative to the Erk waves. This experiment, while not essential for the current findings to have merit, would significantly help our understanding how those two pathways interact.We completely agree. We are working to build a GSK-3β KTR reporter to address this as there are no currently available Wnt reporters that can address cell dynamics. Unfortunately to date, we do not have conclusive experiments that our GSK-3 KTR is faithfully reporting on endogenous GSK-3 activity. We have used a GFP reporter driven by TCF/LEF and found this to respond to Wnt inhibition over multiple days, however the long half-life of GFP prevents accurate dynamics data from being collected within the short time scales of stochastic Erk waves. Adding a degradation sequence to GFP also does not work as the basal fluorescence intensity of GFP is too low for accurate measurements. Therefore, we cannot include the crosstalk between Erk and Wnt dynamics directly at this time. Regardless, we have strengthened the effects of Wnt on Erk signal with our response to question 5 below.5) The -Wnt3a phenotype should be confirmed by using a Wnt3a inhibitor.Agreed. We have added data from experiments using the Wnt inhibitor Pyrvinium. We show that inhibition of Wnt results in activation of Erk and loss of nodes in a dose dependent manner .6) Figure 4B: please add a control image.Control images were added to this figure.


People with advanced cancer need to balance their resources and energy in order to experience enjoyment and quality of life in the time they have left. A resource-oriented intervention is developed targeting these aspects. The present protocol presents a feasibility study of this resource-oriented intervention in people with advanced cancer.A feasibility study with a repeated-measurement design without a control group will be conducted at the research clinic of REHPA, the Danish Knowledge Center for Rehabilitation and Palliative Care. Data will be gathered at baseline, during and after a 5-day residential stay, after 6 weeks, during a 2-day follow-up stay and after 12 weeks. In total, 20–25 home-living adults (≥ 18 years) with advanced cancer reporting needs in everyday life will be recruited. The intervention consists of workshops and engagement in physical and creative everyday activities provided by a multidisciplinary team.Outcome measures are quality of life, physical function and fatigue, which will be assessed using the European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire Core-30. Balance in everyday activities will be assessed using the Occupational Balance Questionnaire.Feasibility data will also be collected regarding (1) fidelity, (2) adherence, (3) dose and (4) reach and mechanisms of impact. For exploration of mechanism of impact, participant observations and focus group interviews will be used.This study presents a new approach in rehabilitation and palliative care aimed at supporting people with advanced cancer; instead of identification of problems, the present resource-oriented palliative rehabilitation intervention will target people’s resources, enhancing balance in everyday activities and underpinning enjoyment and quality of life. The results from the feasibility study can inform ways in which to support the everyday life of people with advanced cancer and thus have potential to improve their quality of life. The long-term perspectives are to evaluate the intervention in terms of effect, process and cost-effectiveness. This will provide evidence to adjust the content of rehabilitation and palliative care for this group of people.NCT04772690Name of the registry: Balance, Activity and Quality of Life (BAL)Date of registry: February 26, 2021 The content, delivery and outcomes of a newly developed resource-oriented intervention for people with advanced cancer should be feasibility tested.The present study presents a protocol for a feasibility study evaluating a resource-oriented intervention.The present feasibility study may contribute with important knowledge to further develop the contents and delivery of a resource-oriented palliative rehabilitation intervention.People with advanced cancer wish to manage everyday activities and experience enjoyment in their everyday lives , 2. FurtResearch shows that people with advanced cancer often suffer from fatigue and find it difficult to prioritise their energy and strength in a manner allowing them to manage activities that are important to them and to achieve a balanced mix of everyday activities , 4, 8. BTo date, neither rehabilitation nor palliative care efforts have focused on ensuring a balanced mix of everyday activities for this group of people or on how enjoyment can be facilitated , 14. OnlAn intervention for people with advanced cancer that targets balance in everyday activities calls for a complex intervention that integrates rehabilitation and palliative care principles by ensuring that the patient’s functional level is maintained while also providing the required relief and support , 23. TheThe present paper outlines the protocol for a feasibility study that will test contents and delivery of a resource-oriented palliative rehabilitation intervention in people with advanced cancer . The folWhich intervention sessions are particularly relevant in the experience of the participants and the healthcare professionals?How do the participants experience, interact and respond to receiving a resource-oriented palliative rehabilitation intervention?Fidelity: Do the healthcare professionals deliver the intervention as planned? And do the healthcare professionals find that they have sufficient knowledge about the intervention?Adherence: Are the participants able to participate in the intervention ?Dose: Which sessions were offered and what was the overall time expenditure?Reach: What characterises the group that received the intervention?Fidelity, adherence, dose and reach of version 1.0 of the intervention manualAre changes observed in the participant’s quality of life, balance in everyday activities, physical function and fatigue after having received a resource-oriented palliative rehabilitation intervention?5)Which outcomes were not completed sufficiently (missing data)?A feasibility study with a repeated-measurement design without a control group will be conducted at the research clinic of REHPA, the Knowledge Center for Rehabilitation and Palliation . REHPA is part of Odense University Hospital, Denmark. The REHPA research clinic offers residential intervention stays for people with life-threatening illness. A residential intervention stay typically consists of a 5-day stay followed by a 2-day follow-up stay approximately 12 weeks later. Since the feasibility study is to be conducted at the REHPA research clinic, a similar structure for the intervention informed the present study. We also know from existing research that 12 weeks of intervention are appropriate for people with advanced cancer since they have a shorter life expectancy , 17, 18.Adult (≥ 18 years) and residing in his or her own homeHas chronic or advanced cancerExperiences a need for support to manage everyday activities and to enhance the balance between various activities and tasks of daily living, e.g. enhance the balance between necessary activities and activities that produce enjoyment and meaningfulnessIs able to participate in the course and willing to complete questionnaires and participate in interviewsMust be independent with respect to personal activities of daily living Speaks and understands Danish languageParticipants need to meet the following inclusion criteria:REHPA will prepare participant information, which will be sent to all palliative teams at hospitals, relevant municipalities, patient associations and cancer counselling services. In addition, the participant information will also be made available on REHPA’s web site and social media like LinkedIn and Facebook. The general practitioner or oncologist at the hospitals will assess potential participants based on the above-listed inclusion and exclusion criteria and will refer them to a REHPA stay if they fulfil these criteria. A responsible clinical healthcare worker will then in collaboration with the group of researchers decide who is eligible for a stay. Potential participants will receive detailed verbal and written information about the study. Prior to inclusion, the study participants are to provide written informed consent.The “template for intervention description and replication” (TIDieR) checklist will be used to describe the intervention . The intIntroduction to activity, balance and everyday lifeIntroduction to “Walk to get happy”— activities in natureMy everyday routine and activities: introduction to diariesBalancing resources, fatigue and energy — how to?My everyday life — balance, challenges and enjoyment“Walk to get happy” — activities in natureLife in movementYogaMeaningful activities: what makes you happy?Creative expressionRelaxing massage: “be good to yourself”Values and action plan“Developments since the previous session”Life in movement — family, friends and networkBody and movementThe intervention will target peoples’ resources, enhance balance in everyday activities and underpin enjoyment and quality of life. The intervention is structured much like standard REHPA stays with a combination of group presentations and activities . In ordeThus, a total of 15 sessions are planned during the 5-day stay and 2-day follow-up stay combined. Each session has a duration of 45–150 min. Additionally, two individual conversations are offered at both stays and one optional session during the 2-day stay, the contents of which is determined by the participants. An optional half-way follow-up telephone contact is also offered to the participants. The sessions are imparted by a multidisciplinary team consisting of a nurse, an occupational therapist, a physiotherapist, a medical trained artist, a mindfulness coach and a social worker. The intervention is primarily group-based, and all 15 sessions are mandatory , a café area and several group rooms. REHPA is located in a recreational area characterised by natural beauty and easy access to walking or running, etc. by the ramparts of Nyborg Castle .Data will be collected at baseline (T1), during the 5-day residential stay (T2), after the 5-day stay has concluded (T3), before the follow-up stay at 6 weeks (T4), during the follow-up stay (T5) and after 12 weeks of follow-up (T6). Data at T2 and T5 is collected after each session for all participants. The following data collection methods will be used: (1) questionnaires, (2) focus group interviews and (3) participant observations.Since the intervention aims to enhance the participants’ quality of life and balance in everyday activities through prioritisation of energy and strength, engagement in physical and meaningful and joyful activities, the following outcomes will be chosen: (1) quality of life, (2) balance in everyday activity, (3) physical function and (4) fatigue, collected at T1, T3, T4 and T6. The outcome questionnaires will be sent and filled out electronically by the participants using the Civil Registration System in Denmark and the Research Electronic Data Capture (REDCaP). Two written reminders will be sent 3 and 6 days after deadline. Then, a reminder will be conducted by phone.Quality of life, physical function and fatigue are measured using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core-30 (EORTC QLQ-C-30). The EORTC QLQ-C-30 is a cancer-specific questionnaire containing 30 questions that address function as well as symptoms and quality of life. Answers are scored on an ordinal scale ranging from 1 to 4 . Additionally, the questionnaire measures quality of life using an ordinal scale ranging from 1 to 7. Subsequently, the ordinal data are transformed into a score ranging from 0 to 100, where a higher score equals better function and higher quality of life or poorer experienced symptoms. The EORTC QLQ-C-30 was found to be valid, reliable and associated with high response rates among persons with advanced cancer .The Occupational Balance Questionnaire (OBQ) is a generic questionnaire comprising a total of 11 items that provide an overall assessment of balance in everyday activities. The items assess in different ways the satisfaction with the amount of and variation in various everyday activities in which people become engaged. Each question is scored on a 4-step ordinal scale ranging from 1 = completely disagree to 4 = completely agree. Based on the 11 questions, a sum score ranging from 11 to 44 is calculated. A higher score indicates a better balance in everyday activities. The QBQ has be found to be valid and reliable , 38.An intervention logbook will be prepared that collects data about: (1) dose (duration and number of intervention sessions given) (T2 and T5), (2) if the contents were delivered as planned (fidelity) (T2 and T5), 3) if the participants were able to participate in the intervention sessions (adherence) (T2 and T5) and (4) the relevance of the intervention sessions (T2 and T5). The intervention logbook will be developed based on the guidance by O’Cathain et al. about feasibility studies . We also if the pData on reach will be collected based on the REHPA’s “How are you?” questionnaire  in whichData on the participants’ experiences, interaction and responses to the intervention will be collected through participant observation and focus group interviews. Participant observation is conducted during the two stays to gain insight into the participants’ immediate reactions to and experience with the intervention and their interaction with the REHPA healthcare professionals. Furthermore, a structured observation table will be used to register expressions of flow and enjoyment while the participants are engaged in session 10. Focus group interviews will be conducted on the 5th day of the 5-day stay and again at the second stay in smaller 5–7 participant groups .A focus group interview will be conducted with the healthcare professionals who imparted the intervention in order to collect their experiences and perceptions from this type of intervention .There are no requirements as to the number of participants needed in feasibility studies . We deemContinuous, normally distributed data are analysed by mean and standard deviation (SD). Otherwise, medians and percentiles will be used. Ordinal data are analysed by medians and percentiles, whereas categorical data and dichotomous data are analysed using numbers and percentages. The number of missing answers in the outcome instruments is calculated and presented descriptively as numbers and percentages. Wilcoxon signed-rank test will be used to analyse changes from T1-T3, T1-T4, and again from T1-T6 with respect to quality of life, balance in everyday activities, physical function and fatigue. Furthermore, a responder analysis will be conducted to determine how many of the participants have achieved a clinically relevant change (5–10 points) . This anQualitative data will be transcribed verbatim from interviews and field notes and analysed using thematic analysis . First, The study follows the principles of the Helsinki Declaration . The sciThis protocol for a feasibility study presents a new approach in rehabilitation and palliative care for supporting people with advanced cancer. Instead of a predominant focus on functional problems and suffering, the present resource-oriented palliative rehabilitation intervention will target the participants’ resources, enhance balance in everyday activities and underpin experiences of enjoyment. In particular, leisure activities seem to be bring enjoyment for people with advanced cancer while at the same time they can support functioning . It is tThe MRC guide recommends drawing on both clinical expertise and evidence when developing a new intervention . The eviSince the intervention is newly developed, several uncertainties remain. These uncertainties need to be tested and explored in a feasibility study before proceeding to a pilot and RCT study . The pre


This is an exciting moment for researchers and clinicians involved in the treatment of Squamous Cell Carcinoma of the Anus (SCCA). Since the consolidation of the chemoradiotherapy with mitomycin and fluoropyrimidine as a standard treatment for localized disease, no clear advances have been achieved for several decades, and the disease remained out of the focus of the development of breakthrough strategies, partly due to its low incidence, especially at advanced stages. However, in last years, well-designed prospective academic and industrial trials are surging, as well as numerous biological and translational studies to better understand this orphanage cancer.Recent data consolidated taxane-based doublet or triplet chemotherapies as standard treatments in front line for metastatic or non-resectable locally advanced diseases, demonstrated the interest of PD1/L1 inhibitors in a subgroup of patients in chemorefractory situation, and also validated the liquid biopsy for HPV oncoproteins (HPV ctDNA) as the best prognostic tool with high sensitivity and specificity in localized and advanced stages.In this collection entitled “New understanding and researches in SCCA”, 8 articles enrich the present knowledge in SCCA and highlight the future directions in its treatment strategies. We believe that these publications will help clinicians in their daily practice, and give an insight in current status of research in SCCA.Three ongoing prospective trials are presented. One in locally advanced stage, one in first-line metastatic disease, and one in chemorefractory patients.Rebucci-Peixoto et al.). Telomerase is an enzyme that is overexpressed in more than 90% of human cancers, and thus prevent the tumor senescence conferring a form of immortality. Previous data have shown that anti-telomerase immunity of the patients was correlated to survival in different cancers, including SCCA  presented here is a phase II clinical trial aiming at improving the anti-PD1/PDL1 efficacy by using a therapeutic cancer vaccine composed of two peptides derived from telomerase (UCPVax), in HPV-related cancers including SCCA . Retifanlimab has already demonstrated its safety and efficacy in SCCA patients in PODIUM 202 phase II trial in chemorefractory patients  . RecentlKim et al.). This is a logical step considering the 100% of disease control rate in chemo-naïve advanced SCCA patients (29/115) treated with DCF regimen, with 90% of ORR including 55% of CRR, and almost 25% of disease-free survival at 5 years in favor of its effectiveness in micrometastases  in patients with stage III SCCA disease (N+ or T4 N0) , and 3 patients had synchronous or metachronous metastases. ORR was achieved in 87.5% and CRR was observed in 25% of patients after mDCF. The disease control rate was 100% during chemotherapy. One patient with metachronous liver metastasis underwent to liver surgery after mDCF and a pathological complete response was observed. Other 8 patients received a chemoradiotherapy of the primary tumor, and six of them (75%) presented a complete response, including a pathological complete response in the unique patient that underwent surgery of his primary tumor. After a median follow-up of 33 months, 78% of patients were alive and free of disease, and the median OS was not reached at 6 years. This article confirms the high efficacy of mDCF regimen observed in SCCA and should be considered as a standard in advanced SCCR. It also highlights the importance of complementary management with a majority of patients being alive at 6 years. This live-saving strategy is immediately applicable in the daily practice, and we believe that these patients should be treated in the referent centers by a multidisciplinary team.Squamous-cell carcinoma of the rectum (SCCR) is a very rare situation and its prognosis is poorer than SCCA and adenocarcinoma of the rectum (ACR). The 5-years OS rate is about 30% for a stage III disease (compared to 65% for SCCA and ACR) and less than 10% for a stage IV disease (compared to 20% for ACR and 30% for SCCA). In this collection, Looking at the radiation oncology world, 2 retrospective studies are presented in this collection, one of them dealing with the role of modern irradiation techniques and with MRI-based radiomics in SCCA; and one exploring the role of potentially curative local chemoradiotherapy in metastatic SCCA patients.Patterns of treatment for SCCA have changed over the time in the last decades. Indeed, we understood the detrimental impact of a longer overall treatment time, the explored different chemotherapy schedules, and we implemented newer and more conformal radiotherapy techniques.Bonù et al. presented in this collection, the authors showed the positive impact of the modern RT techniques. They showed a reduction of the rate of severe acute toxicity with the implementation of Intensity Modulated Radiotherapy (IMRT), and an improvement of the local control, colostomy free survival and overall survival rates, probably related both to the optimization of the treatments schedules, but also to the introduction of the Image Guided Radiotherapy (IGRT), allowing a better targeting of the treatment volumes. These data confirm the positive impact of the IMRT already showed by the phase II RTOG 0529 trial published in 2013  in radiotherapy in SCCA patients.Radiomics and radiogenomics are being tested in clinical oncology, and it has been already showed that radiomics analyses could predict the clinical behavior of several gastrointestinal malignancies. The rarity of SCCA is a major limiting factor to realize such a kind of studies in this clinical setting. This is the case also for the data presented by Grave et al.). Here, 16 SCCA patients treated with DCF polychemotherapy in the context of 3 prospective trials were selected. All these patients achieved an objective response to chemotherapy and were treated by a chemoradiotherapy on their primary tumor site. After a median follow-up of 42 months, the authors showed, in this very selected group of patients, high rates of complete local response, comparable to those usually reported in non-metastatic patients. Noteworthy, these results were obtained despite interruptions due to toxicities and low compliance to concomitant chemotherapy . This is the first report, based on prospective data, exploring the role of pelvic chemoradiotherapy in metastatic SCCA patients.The second article explored the role of local treatments as a consolidation therapy in advanced SCCA patients exposed to DCF polychemotherapy . For this, they measured the expression of these 2 markers by fluorescence immunohistochemistry in 63 SSCA patients whose tumor samples were available. Different thresholds were studied, and the data showed that a threshold for tumor PD-L1 expression of ≥5% was associated with better overall survival, which is not the case with the currently used threshold of 1%. If the CD8 expression did not influence the overall survival, the combination of these 2 markers showed that overall survival was driven by tumor PD-L1 expression but may be influenced by CD8 infiltration levels. The authors showed that a threshold for PD-L1 expression (tumor PD-L1 ≥5%) was a prognostic biomarker associated with overall survival in SCCA patients treated by chemoradiotherapy. While the prognostic value of this new threshold needs to be validated in future trials, these data suggest that tumor PD-L1 ≥5% may predict responses to immunotherapies in SCCA patients.PD-L1 expression has been shown to be prognostic in many cancers and used in consideration of checkpoint inhibitor immunotherapy. Nevertheless, the threshold of PD-L1 expression positivity currently used in SCCA patients may not be optimal to select patients who could benefit from the immunotherapy. This hypothesis was studied by Chan et al. who evaluated the prognostic value of PD-L1 and CD8 expression in SCCA patients treated by chemoradiotherapy (These results demonstrate the interest of identifying and validating the SCCA-related predictive biomarkers. These biomarkers will allow a better stratification of SCCA patients and will limit the risks of recurrence by adapting treatments in these patients.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.