Cic from the mouse dentate gyrus compromises the adult neural progenitor cell pool without altering their proliferative potential. We further demonstrate that the loss of Cic impedes neuronal lineage development and disrupts dendritic arborization and migration of adult-born neurons. Our study uncovers a previously unrecognized role of CIC in neurogenesis of the adult dentate gyrus.New neurons continuously arise from neural progenitor cells in the dentate gyrus of the adult hippocampus to support ongoing learning and memory formation. To generate functional adult-born neurons, neural progenitor cells proliferate to expand the precursor cell pool and differentiate into neurons. Newly generated cells then undergo postmitotic maturation to migrate to their final destination and develop elaborate dendritic branching, which allows them to receive input signals. Little is known about factors that regulate neuronal differentiation, migration, and dendrite maturation during adult hippocampal neurogenesis. Here, we show that the transcriptional repressor protein capicua (CIC) exhibits dynamic expression in the adult dentate gyrus. Conditional deletion of  Quiescent radial glial-like (RGL) neural progenitor cells, which reside in the subgranular zone (SGZ) of the hippocampal dentate gyrus, develop into mature granule neurons through four phases: a progenitor cell phase, an early survival phase, a postmitotic maturation phase, and a late survival/maturation phase2. This entire process takes about 6–8 weeks and encompasses six developmental milestones which can be identified based on molecular markers and cell morphology  Cic knockout mice die by weaning age with prominent maturation defects in cortical neurons and dentate gyrus granule neurons11. Neocortical excitatory neurons in the Emx1-Cre Cic knockout mice, which lack Cic in forebrain excitatory neurons and glia, have reduced post-mitotic survival and the surviving neurons develop abnormal dendritic arborization12. These findings highlight the importance of CIC in neuronal maturation and dendrite development during early postnatal brain development. However, whether CIC is similarly required for neurons to mature during adult neurogenesis remains unknown.To further investigate this possibility, we turned to capicua (CIC), a transcriptional repressor first identified in To understand the role of CIC in the adult brain, we evaluated CIC expression and its role in adult hippocampal neurogenesis. We find that CIC is required for maintenance of the adult progenitor cell pool, proper neuronal lineage development, as well as development of dendritic branching of new neurons. Loss of CIC also disrupted normal migration of some adult-born neurons. Our study uncovers a previously unrecognized role of CIC in adult hippocampal neurogenesis and increases our knowledge of transcription factors that regulate adult-born neuron development in the dentate gyrus.2. IPCs give rise to neuroblasts that eventually exit the cell cycle to become immature and then mature granule neurons that localize to the granule cell layer13. To determine which of these cell types express CIC, we co-immunostained for CIC and two specific markers, SOX2 and DCX, which labelled two broad, largely non-overlapping cell stages in the adult dentate gyrus  . As DCX+ cells progressed to mature granule neurons, which were classified as cells in the granular layer negative for SOX2 and DCX, CIC was re-expressed and reached the highest levels in the nucleus , where neurogenesis takes place. There are several cell types contained in the dentate gyrus . The Emx1-Cre allele expresses the cre recombinase in forebrain neural progenitor cells starting from embryonic day 9.5, resulting in cre-mediated recombination in the developing and adult dentate gyrus14. In the present study, we investigated cellular defects of the dentate gyrus in the Cicflox/flox (control), the Emx1-Cre; Cicflox/+ , and the Emx1-Cre; Cicflox/flox  mice. We first tested whether cre-mediated recombination in the adult dentate gyrus was complete in the Emx1-Cre Cic knockout mice. Immunostaining for CIC in the control and knockout mice showed that CIC was efficiently deleted from Emx1-lineage cells in all of the adult neurogenic cell stages, which included SOX2+ neural progenitor cells, DCX+ cells and mature granule neurons . CIC+ cells were occasionally found in the adult dentate gyrus of the knockout mice in what were likely non-Emx1-lineage cells.To examine the function of CIC in adult hippocampal neurogenesis, we used the Emx1-Cre driver is turned on during early embryonic development, we first determined whether Cic deletion in these mice affected the formation of the adult neurogenic SGZ. We defined the SGZ as “a layer of cells expanding 5 μm into the hilus and 15 μm into the granular cell layer” as previously described15. Our analysis of 11-week-old control and Emx1-Cre Cic knockout mice showed that the size of the SGZ was not significantly altered in the knockout mice . We next assessed the effects of Cic deletion on neural progenitor cell proliferation. To this end, we used EdU (5-ethynyl-2′-deoxyuridine), a thymidine analogue, to label cells that had entered the S phase of the cell cycle during a 4-h period. The length of S phase for SGZ neural progenitor cells is 2–2.5 h and the total cell cycle length is about 22–24 h16. Therefore, this experimental paradigm labelled only those cells that had recently entered, and remained in, the cell cycle. We found a decrease in the number of EdU+ cells in the SGZ of the Emx1-Cre Cic knockout mice compared to the control mice . We next quantified the cells that were co-labelled with SOX2 and the proliferation marker Ki67, and found that fewer Ki67+ SOX2+ cells were present in the knockout mice . We did not detect any differences in cleaved CASP3+ cells among the three genotypes . However, it needs to be recognized that CASP3 activation is transient and that CASP3-independent programmed cell death pathways have been shown to regulate adult neurogenesis23. We next injected EdU into the mice (two injections that were two hours apart) and analysed them three weeks after injections . Indeed, we also observed some TBR2+ IPCs abnormally located to the molecular layer of the Emx1-Cre Cic knockout mice , further demonstrating aberrant migration of immature neuronal lineage cells in the knockout mice.We next asked whether loss of CIC had any effect on cell migration. During adult hippocampal neurogenesis, most migration occurs in DCX-expressing cell stages+ cells, concomitant with uncontrolled radial migration of some of these cells.In brief, CIC regulates dendritic development of immature neurons in the adult dentate gyrus. Loss of CIC impairs dendritic arborization of DCX5. As CIC regulates the expression of other transcription factors involved in cell growth and differentiation29, we hypothesized that deletion of CIC from the adult dentate gyrus disrupted downstream transcription factor networks. We reasoned that a transcription factor repressed by CIC would have an expression pattern inversely correlated with the expression of CIC along the neurogenic lineage. To identify such factors, we took a list of CIC-bound transcription factor genes in neural stem cells and looked for ones with low expression in neural progenitor cells and mature neurons but high expression in neuroblasts/immature neurons using a single-cell RNA sequencing dataset of the adult dentate gyrus30. This analysis revealed only two candidates, Nfia and Nfib, which are both members of the nuclear factor I (NFI) family .Adult hippocampal neurogenesis is driven by a coordinated network of transcription factors that orchestrate the developmental sequence of proliferation, differentiation and maturation+ cells, with NFIB experiencing a greater increase in expression from the transition of SOX2+ neural progenitor cells to DCX+ cells . Moreover, within the DCX+ population, strong NFIB expression was seen in majority of the cells except the ones with the most mature morphology, namely those with an elaborate dendritic tree, an enlarged cell body and low DCX expression32. As DCX+ cells developed into mature neurons, NFIA and NFIB levels dropped. Thus, expression of NFIA and NFIB along the granule cell lineage development inversely correlates with the expression of CIC .By co-staining NFIA and NFIB with cell-stage specific markers, we confirmed that both NFIA and NFIB were most highly expressed in DCXEmx1-Cre Cic knockout mice. While deletion of CIC did not lead to ectopic NFIA or NFIB expression in neural progenitor cells or mature neurons, NFIB (but not NFIA) levels were significantly upregulated in DCX+ cells of the Emx1-Cre Cic knockout mice compared with the control mice , we then asked whether increased levels of NFIB in the Emx1-Cre Cic knockout mice would alter the development of DCX+ cells. In the control mice, about 50% of DCX+ cells expressed NFIB at levels higher than those in SOX2+ progenitor cells; these NFIBhi DCX+ cells exhibited a relatively immature morphology compared to DCX+ cells with low NFIB expression. In the knockout mice, however, roughly 75% of DCX+ cells co-expressed high levels of NFIB , this result indicates an expansion of the less mature DCX+ subgroups in the knockout mice. Since the total number of DCX+ cells did not differ between the different lines of mice  Deletion of CIC from the dentate gyrus reduces the adult progenitor cell pool without affecting the proliferation potential of progenitor cells. (2) Despite the reduction in the progenitor cell pool, Cic deletion does not alter the number of DCX+ cells. This is likely due to altered differentiation progression, as we did not find an apparent effect on cell death of the DCX+ population. (3) Loss of CIC impedes dendritic development and migration of maturing neurons. Our findings provide evidence for an important role of CIC in adult hippocampal neurogenesis.In the present study, we demonstrate that CIC is dynamically regulated during neuronal lineage progression in the adult dentate gyrus. Using the 30. It is widely expressed in the embryonic and adult brains12. Within neurogenic regions, CIC expression is low in neural stem and progenitor cells but high in fully differentiated neurons33. However, whether CIC upregulation during neurogenesis is strictly linear or has additional dynamics is unknown. Here, by comparing CIC expression in different cell stages, we made a surprising discovery that CIC is first downregulated in DCX+ cells and then upregulated as new neurons exit the DCX-expressing stage. The dip in CIC levels in DCX+ cells may allow for the onset of a transcriptional program that is uniquely required for the differentiation and/or survival of these cells. This notion is supported by the results of our EdU-labelling study showing increased percentage of EdU+ DCX+ cells in the knockout mice, which suggests increased DCX+ cell survival and/or increased progenitor cell differentiation into DCX+ cells upon the loss of CIC. New neurons begin to express DCX when they start to migrate and grow their dendrites22. These specialized behaviours must be supported by a set of genes that promote cell motility and proper neurite outgrowth. CIC may regulate some of these genes as we observed impaired dendritic development and migration of DCX+ cells when we deleted CIC from the dentate gyrus. What mediates the dynamic expression of CIC during neurogenesis remains to be investigated, but CIC is known to be negatively regulated by the growth factor–RAS/MAPK signalling pathway29. Therefore, CIC downregulation in DCX+ cells may serve to integrate external neurogenic cues, such as growth factor signalling, to neuronal lineage development by enabling appropriate activation of genes involved in survival and/or neuronal differentiation, cell motility and dendritic outgrowth.CIC is a transcriptional repressor that recruits other co-repressor proteins, such as the mSWI/SNF and SIN3-HDAC complexes, to repress target gene expression10. Surprisingly, we did not find a change in the proliferation potential of SOX2+ progenitor cells when CIC was deleted from the dentate gyrus. Our observation is in agreement with a previous study using the Emx1-Cre Cic knockout mice to demonstrate that loss of CIC does not alter the proliferation rate of embryonic ventricular zone neural progenitor cells12. On the other hand, deletion of CIC increases proliferation of cultured neural progenitor cells isolated from the embryonic ventricular zone and perinatal subventricular zone33. Such discrepancy may arise from the use of in vivo versus in vitro systems. It is possible that when taken out of the neurogenic microenvironment and cultured in vitro, Cic-null neural stem cells sustain cell proliferation via a cell-autonomous mechanism. In vivo, however, proliferation of progenitor cells is regulated by a plethora of extracellular signals in the neurogenic niche5. Diverse regulatory feedback signals might keep the proliferation capacity of Cic knockout progenitor cells in check to prevent precocious stem cell pool exhaustion. Although the proliferation potential of neural progenitor cells is not changed, the progenitor cell pool is reduced in the Emx1-Cre Cic knockout mice. This could be due to increased progenitor cell differentiation into DCX+ cells as our data suggest. Alternatively, there might be a defect in the initial establishment of adult neural progenitors in the knockout dentate gyrus. Future studies are needed to determine whether CIC plays a role in the early postnatal dentate gyrus to establish adult neural progenitors.As a gatekeeper of the RAS/MAPK pathway, CIC restricts cell proliferation in the contexts of development and cancer+ cell development. This is supported by (1) EdU-labelling study showing impaired progression of knockout DCX+ cells into CALB1+ mature neurons, (2) morphological analysis revealing reduced dendritic complexity in knockout DCX+ cells, and (3) molecular analysis demonstrating an expansion of DCX+ cells co-expressing high levels of NFIB, which marks the less mature subgroups. Previous studies have demonstrated a role for CIC in dendrite development and neuronal maturation during early brain development. Deletion of CIC reduces dendritic arborization of layers 2–4 neurons in the cerebral cortex during early postnatal development12. Therefore, CIC may regulate dendritic development across different neuronal subtypes. Moreover, deletion of CIC using the pan-neural Nestin-Cre driver expands the pool of neuroblasts with a less mature phenotype in the early postnatal (2-week-old) dentate gyrus11. CIC thus seems to play a consistent role in dentate gyrus neurogenesis whether in early postnatal or adult periods. In addition to highlighting the requirement of CIC for dendritic development of DCX+ cells, our data suggest that CIC also plays a role in the regulation of migration of these cells. DCX + cells normally do not migrate pass the granule cell layer27. However, we consistently observed DCX+ cells abnormally located in the molecular layer of the Emx1-Cre Cic knockout mice. Notably, only a small fraction of DCX+ cells in the Cic knockout mice exhibit abnormal migration. This might be due to other redundant/compensatory pathways that safeguard global neuronal migration in the absence of CIC. As little is known about the molecular mechanisms underlying neuronal migration in the adult dentate gyrus, future studies that aim to capture these abnormally migrated DCX+ cells and interrogate their molecular signatures using single-cell approaches may yield new insight into the regulation of migration of adult-born neurons.A striking phenotype uncovered in the absence of CIC is the disruption of DCX+ cell stage, a pattern similar to that of NFIX13. While NFIX has been shown to be required for neuroblast maturation and dendritic development, the role of NFIA and NFIB in the adult dentate gyrus needs to be investigated. Much of our knowledge of the NFI factors in normal brain development comes from loss-of-function studies34. The consequences of ectopic or overexpression of these factors remain largely unexplored. Nonetheless, evidence suggests that the brain is highly sensitive to the dosage of NFI factors. Astrocyte differentiation is reduced and delayed as a result of NFIB loss, while precocious astrogliosis is observed upon ectopic expression of NFIB37. Adult hippocampal neurogenesis is biased toward an oligodendrocyte cell fate by NFIX deletion or NFIB overexpression in neural stem cells38. Importantly, inhibition of NFI factors in developing cerebellar granule neurons impedes neuronal process formation and migration via downregulation of cell adhesion molecules39. Our finding that upregulation of NFIB in CIC knockout DCX+ cells is associated with abnormal dendritic development and migration further attests to the importance of NFI factors in neuronal maturation, and raises the possibility that proper neuronal migration and maturation requires an optimal dosage of NFI factors. Overexpression of NFI factors might lead to increased expression of cell adhesion molecules, disrupting the normal migratory behaviour of DCX+ cells. Interestingly, we observe upregulation only of NFIB and not NFIA in the Emx1-Cre Cic knockout mice, suggesting differential regulation of the two factors by CIC or compensatory mechanisms that maintain the steady-state levels of NFIA in the absence of CIC. The NFI factors have very similar expression patterns during brain development and overlapping transcriptional targets41, but how they might be regulated differently in diverse cellular contexts is not well understood. Our results open the door for future investigative studies to dissect the mechanisms by which NFI factors are differentially modulated in the adult neurogenic lineage. Moreover, it is intriguing that loss of CIC only increased NFIB levels in DCX+ cells, where CIC is normally kept at a very low level. Future studies are required to test if CIC directly regulates Nifb and whether upregulation of NFIB is a cause or consequence of altered development of DCX+ cells.The nuclear factor I (NFI) family of transcription factors are important players in brain development. Our results demonstrate that within the adult hippocampal neurogenic lineage, NFIA and NFIB both have peak expression at the DCXEmx1-Cre Cic knockout mice, it needs to be recognized that the observed defects may partly arise from Cic deletion in the developing brain, as the cre recombinase is turned on in forebrain progenitors from E9.514. However, it is worth noting that the Emx1-Cre Cic knockout mice have a grossly normal dentate gyrus with similar cellularity to the control mice at 5 weeks of age12. We also did not find a significant difference in the size of the neurogenic SGZ in 11-week-old mice. Nonetheless, loss of Cic in mature granule neurons in the Emx1-Cre Cic knockout mice may change the local environment of adult neurogenesis, contributing to the observed defects in a non-cell autonomous manner. Therefore, establishing a mechanistic or a causative link will required further studies using knockout mice with conditional Cic inactivation in adult neural stem cells (such as Cic deletion using the Nes-Cre/ERT2 driver line42).Although our present data reveal several abnormalities of adult hippocampal neurogenesis in the + cells . The highly branched dendrites of DCX+ immature neurons provide the anatomical basis for receiving information from the entorhinal cortex, enabling ongoing learning in the adult brain44. Therefore, dendritic arborization defects in these cells could contribute to the previously observed learning and memory deficits in the Emx1-Cre Cic knockout mice12. Moreover, heterozygous loss-of-function mutations in CIC cause a rare neurodevelopmental syndrome characterized by prominent intellectual disability/learning difficulties in affected individuals45. Given that these pathologies persist into adulthood, our finding that CIC is required for adult hippocampal neurogenesis sets the stage for future investigative studies to understand whether the loss of CIC triggers ongoing learning difficulties in adult patients by impairing adult neurogenesis. Beyond the CIC haploinsufficiency syndrome, CIC may also play a broader role in age-related cognitive decline, as impaired adult hippocampal neurogenesis is believed to contribute to age-associated cognitive deficits48. Aging leads to a reduction in neural progenitor cells51 and a delay in morphological maturation of adult-born granule neurons52. Both of these defects were observed in the Emx1-Cre Cic knockout mice. In future studies, it will be interesting to determine whether CIC expression along the adult neurogenic lineage is altered by aging, and whether aging exacerbates the neurogenesis defects of the Emx1-Cre Cic knockout mice.In conclusion, our present study identifies CIC as a critical regulator of adult dentate gyrus neurogenesis and highlights the requirement of CIC for proper dendritic development of DCXCicflox mice has been previously described12. The Cicflox mice are also available from The Jackson Laboratory (stock number: 030555). Wildtype C57BL/6J mice and Emx1-Cre mice  were obtained from The Jackson Laboratory. No sex differences were observed in our studies. Both male and female mice were used for experiments. For studies involving wildtype C57BL/6J animals, animals were eight weeks of age. For EdU labelling experiments, 8-week-old mice were used. For all other experiments, 11- to 20-week old Emx1-Cre; Cicflox/flox mice were used, and their littermate Cicflox/flox and Emx1-Cre; Cicflox/+ mice were used as controls.Generation of the https://arriveguidelines.org.All procedures in mice were approved by the Animal Care and Use Committee of the University of Alberta. All methods were performed in accordance with the relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines: EdU  was reconstituted in DMSO to a concentration of 100 mg/mL, aliquoted and stored at − 20 °C until further use. For progenitor proliferation analysis, on the day of experiment, EdU was diluted with endotoxin-free PBS to a final concentration of 5 mg/ml, and injected intraperitoneally into 8-week-old mice to a final in vivo concentration of 50 µg/g body weight. A second dose of EdU was injected two hours after the first injection to maximize labelling efficiency. Mice were euthanized two hours or three weeks after the second injection by an intraperitoneal injection of sodium pentobarbital and used for brain section preparation.Mice were deeply anaesthetized via intraperitoneal injection of sodium pentobarbital, and transcardially perfused first with PBS then with 4% paraformaldehyde in PBS. Mouse brains were then dissected and immersed in a 4% paraformaldehyde solution overnight at 4 °C to further fix the tissues, followed by submerging the brains in cryoprotective 15% and 30% sucrose, each for 24 h. The mouse brains were cut coronally using a brain matrix and cryo-embedded using Tissue-Tek OCT compound. Coronal brain sections (40 μm-thick) were cut using a cryostat , transferred to ColorFrost Plus Microscope Slides , and air-dried for immunofluorescence staining. Excess slides were stored at − 80 °C. Frozen slides were thawed at room temperature and dried for several hours to overnight before undergoing staining procedures.EdU detection was performed by using the Click-iT Alexa Fluor 555 dye EdU kit  according to the manufacturer’s protocol. Briefly, slides with brain sections were post-fixed with 4% paraformaldehyde, washed with 3% bovine serum albumin in PBS, then permeabilized with 0.5% Triton-X 100 in PBS at room temperature for 20 min. Slides were then incubated with the Click-iT reaction cocktail that contained Click-iT reaction buffer, Alexa Fluor 555 Azide, CuSO4, and reaction buffer additive for 30 min while being protected from light. Slides were then washed once with 3% BSA in PBS and then three times with PBS before being further processed for immunofluorescence staining or counterstaining with DAPI.12. Slides containing brain sections were first post-fixed in 10% neutral buffered formalin  for 5–10 min, then washed three times with PBS. Antigen retrieval was performed using the Antigen Unmasking Solution  at 95 °C for 20–60 min in a water bath with gentle shaking (60 rpm). Slides were cooled to room temperature and permeabilized with 0.3% Triton X-100 in PBS, then blocked with 5% normal donkey serum in 0.3% Triton X-100 in PBS. Slides were incubated at 4 °C overnight with the primary antibodies in blocking buffer. The slides were washed three times with 0.3% Triton X-100 in PBS, then incubated with secondary antibodies in blocking buffer at room temperature for two hours. The slides were then washed twice with 0.3% Triton X-100 in PBS, and then once with PBS. Autofluorescence quenching was carried out to reduce background autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit  and all slides within one experiment were treated similarly. Slides were counterstained with DAPI and then mounted using VECTASHIELD Vibrance mounting media .Immunofluorescence staining was performed as previously described with modificationsThe following primary antibodies were used for immunofluorescence staining: rabbit anti-Ki67 , rabbit anti-CIC , mouse anti-DCX , goat anti-SOX2 , mouse anti-NES , rabbit anti-TBR2 , rabbit anti CALB1 , rabbit anti-NeuN , rabbit anti-GFAP , rabbit anti-OLIG2 , rabbit anti-NFIB , and rabbit anti-NFIA . The secondary antibodies used were donkey anti-rabbit Alexa Fluor 488 , donkey anti-mouse Alexa Fluor 647 , and donkey anti-goat Alexa Fluor 555 .+ and SOX2+ Ki67+ cells were restricted to cells within the subgranular zone of the dentate gyrus, which was defined as “a layer of cells expanding 5 μm into the hilus and 15 μm into the granular cell layer” as previously described15. Dendrite analyses were carried out using the Simple Neurite Tracer plugin of ImageJ. To quantify NFIA and NFIB immunofluorescence intensity and the number of cells expressing high levels of these proteins, images of single fluorescence channel were adjusted using the MaxEntropy method and an automated threshold calculated by ImageJ. The images were further subjected to Watershed binary processing to split cell clusters into individual cells. Particle analyses were then performed on the processed images, which yielded the number of cells expressing NFIA or NFIB and the mean fluorescence intensity of these cells. Statistical analyses were performed using GraphPad Prism. Detailed information on statistical analyses is given in the figure legends.Immunofluorescent images were taken using a laser-scanning confocal microscope (Zeiss LSM 700). For each animal, tiled and z-stacked images of the dentate gyrus were acquired from at least three comparable coronal sections spanning the entire dorsal dentate gyrus and analyses were performed on images of these sections. Each data point represents the average value from multiple sections per animal. Quantifications of fluorescence intensity and cell counts were carried out using ImageJ. Cell counts for total SOX2Supplementary Information.


Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer.Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC).Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors.Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation. Microbubbles can enhance sonoporation effects on cellular membranes caused by physical forces  from their oscillation or destruction in the acoustic field. Microbubble-assisted ultrasound techniques were successfully applied in several Apart from applications for the treatment of brain tumors, a combination of chemotherapeutic agents and microbubble-assisted ultrasound (sonochemotherapy) has been used in tumors outside the brain. In this regard, Yousefian and colleagues showed a tumor growth delay of murine breast cancer after a combination of bleomycin injection and microbubble-assisted ultrasound, compared to untreated, bleomycin alone or ultrasound alone treated tumors These findings could be especially interesting for patients diagnosed with desmoplastic tumors  that have a poor prognosis. With more than two million cases in 2018, breast cancer is the second most common cancer worldwide One reason for the poor prognosis is the restriction of cytostatic drug delivery to the tumors. The distribution of drugs in tumor tissues does not only depend on the drugs' physicochemical characteristics but also on the composition of the tumors, e.g., density and assembly of tumor cells, the composition of the extracellular matrix, size of the interstitial space, interstitial fluid pressure, and vascularization In this regard, ultrasound is routinely used as an imaging technique for the breast and applied as a monitoring tool for patients undergoing neoadjuvant treatment of breast cancer. Several clinical studies show the feasibility of contrast-enhanced ultrasound (CEUS) for breast cancer diagnosis Contrast-enhanced (Doppler) ultrasound takes advantage of the physical effects of microbubbles as it aims to generate microbubble oscillations Similarly, but with a therapeutic intention, sonopermeation aims at microbubble oscillation- or destruction-induced opening of biological barriers, such as vascular walls. Most preclinical and clinical sonopermeation studies were performed at a relatively low frequency of 1 - 2 MHz or below Taking this notion into account, we performed a prospective clinical study to investigate whether the repeated application of contrast-enhanced Doppler ultrasound with destruction of microbubbles during chemotherapy infusion influences tumor perfusion to improve the response of breast cancer to neoadjuvant chemotherapy. In this context, super-resolution ultrasound imaging ) 2) + cyclophosphamide  every 2 - 3 weeks, followed by 12 weekly cycles of a combination of paclitaxel  with carboplatin . Treatment schemes of patients with Her2neu positive and hormone receptor positive tumors can be found in the methods part of the The study was approved by the RWTH Aachen University Hospital's ethics board and registered in clinicaltrials.gov (NCT03385200). All patients gave their written consent to participate. A detailed description of the clinical trial can be found in the Before the first chemotherapy cycle, patients were randomly assigned to receive either only standard chemotherapy treatment  or additionally high MI contrast-enhanced Doppler ultrasound .8 microbubbles/ml ® . The contrast agent was slowly injected intravenously in a permanent venous port system, which is always placed into the subclavian vein. Only one central slice of the tumor was imaged at a low MI to reduce the effects of the perfusion measurement on the tumor to a minimum. Perfusion measurements were repeated directly after each chemotherapy cycle.Before each chemotherapy infusion, tumor sizes were measured using the Toshiba Aplio 500 ultrasound scanner (14L5 1005BT transducer) in B-mode. Tumor volumes were calculated using the formula W x H x D x π / 6. Next, tumor perfusion was assessed in non-destructive contrast specific mode  < 0.4, framerate 15/sec, persistence 0, gain and dynamic range were kept constant for all measurements) for 180 seconds by measuring the inflow of 0.5 ml  was applied on the whole tumor during the first 18 minutes of chemotherapy infusion in Doppler mode (80% power) using a Philips iU22 ultrasound system  equipped with an L17-5 transducer working at 7 MHz . During this time, six consecutive slow intravenous injections of 0.5 ml SonoVuePost-processing of the acquired ultrasound images was performed as described previously The motion-corrected contrast mode images were used to localize single microbubbles on a highly resolved grid via gauss-detection The vasculature was reconstructed by tracking microbubbles in consecutive frames. The tracking was performed by the Markov chain Monte Carlo data association algorithm (MCMCDA) because of its robustness in case of complex circumstances, like a low frame rate or crossing microbubble tracks due to a low elevational resolution The German State Office for Nature, Environment and Consumer Protection (LANUV) North Rhine-Westphalia approved all animal experiments. A total of 15 female Balb/cAnNRj mice  were housed in groups of 5 animals on spruce granulate bedding  under specific pathogen-free conditions with a 12 h light/dark cycle in a temperature (20-24 °C) and humidity (45-65%) controlled environment according to the guidelines of the “Federation for Laboratory Science Associations” (FELASA). Standard pellets  and acidified water were offered ad libitum.4 murine syngeneic TNBC cells  in 50 µl RPMI 1640  medium were orthotopically injected into the right inguinal fat pad. Tumor size was assessed daily via caliper. When tumors reached 6 - 8 mm in diameter, mice were randomly allocated to three experimental groups (n = 5 per group). Animals of the first group (CTx) received an intravenous infusion of 50 mg/kg bodyweight carboplatin  in 0.9% sterile saline with an infusion rate of 0.01 ml/min for 10 min in a lateral tail vein. The second group (CTx + MB) was treated with five consecutive intravenous injections of 1 x 105 microbubbles SonoVue® in 25 µl 0.9% sterile saline every 2 min, additionally to carboplatin infusion. Animals of the third group (CTx + high MI CDUS) received the carboplatin infusion and five consecutive intravenous injections of 1 × 105 microbubbles SonoVue® in 25 µl 0.9% sterile saline every 2 min while high MI CDUS was performed using the Philips iU22 ultrasound system . The duration of high MI CDUS was reduced to 10 min to ensure equivalent total ultrasound energy deposition in the smaller murine tumors compared to human tumors. The 2 min interval between microbubble injections was chosen as a compromise between a faster clearance of microbubbles due to the higher heartrate in mice and an even distribution of the injections over the 10 min treatment time. The distance of the transducer surface to the mouse tumors was adjusted to 1.5 cm (representing the mean imaging depth in humans) using ultrasound gel . Additionally, the inflow of 1 × 105 microbubbles SonoVue® in 25 µl 0.9% NaCl was imaged using the Vevo3100  small animal imaging system equipped with the MX250 transducer in contrast mode  to assess the tumor perfusion before and after high MI CDUS.After an acclimatization period of 7 days, 4 x 10 Ricinus Communis-agglutinin I  were intravenously injected to stain perfused vessels in the tumor followed by cervical dislocation to euthanize the animals.After completing the carboplatin infusion, 60 µl rhodamine-labeledMurine tumors were collected and bisected. One half was embedded in Tissue Tek® O.C.T. Compound , the other half was snap-frozen for ICP-MS analysis, and both were stored at -80 °C.For comparison of tumors between mouse and human TNBC, histological specimens of 10 untreated 4T1 tumors grown in the same mouse strain were analyzed. Half was embedded in Tissue Tek O.C.T. compound; the other half was embedded in paraffin. Paraffin-embedded murine tumor samples were cut in 5 µm thick slices and stained for connective tissue. Immunofluorescence staining of tumor vasculature was performed on frozen tissue samples. A detailed description of ICP-MS analyses and histological staining can be found in the Paraffin-embedded human tumor samples were cut in 3 µm thick slices. Staining for tissue morphology (hematoxylin & eosin (H&E)) and connective tissue (trichrome staining) were performed. Furthermore, immunofluorescence staining of tumor vasculature was performed. A detailed description of staining procedures can be found in the All data are presented as the median and interquartile range (interquartile 1 - interquartile 3). Statistical analyses were performed using Graph Pad Prism 9.0 . Differences in tumor size and ultrasound parameters between CTx and CTx + high MI CDUS treated patients at the single time points were tested for statistical significance using the Mann-Whitney test. The number of animals per group was calculated using a power calculation Tumor sizes were comparable between CTx and CTx + high MI CDUS treated patients  could be observed from the fourth cycle of chemotherapy onwards in CTx treated patients, whereas tumors of CTx + high MI CDUS treated patients showed significant changes from the fifth cycle of chemotherapy onwards due to one non-responding tumor Figure C.This tendency towards a delayed response of tumors to chemotherapy after high MI CDUS resulted in the decision to pause the clinical study.Almost fifty percent of the tumors were not detectable by ultrasound at cycle seven of chemotherapy anymore. Therefore, only data from the first six cycles were used to compare CTx, and CTx + high MI CDUS treated patients.th chemotherapy cycle. Furthermore, the PE was comparable between the different tumor types.We first searched for longitudinal effects of high MI CDUS on tumor vascularization by comparing the relative PE of CTx and CTx + high MI CDUS treated patients before Figure A and aftNext, changes in tumor vascularization before and directly after high MI CDUS were investigated by comparing the PE values after each chemotherapy infusion with the respective value acquired before chemotherapy. As indicated in Figure Furthermore, the change in Doppler signals during high MI CDUS was assessed in CTx + high MI CDUS treated patients to investigate the occurrence of immediate effects on the tumor vascularization. The analysis of the Doppler positive tumor area did not reveal a clear trend towards changed perfusion Figure D & E.To identify subtle differences in vascularization between CTx and CTx + high MI CDUS treated tumors, mULM was applied. We first analyzed the effects on rBV. Compared to PE, the rBV is a more robust parameter in detecting tumor vascularization as it is not influenced by the injection speed of microbubbles. In line with PE results, there were no significant alterations in rBV from before to after chemotherapy infusion, no significant changes over the treatment period, and comparable values were obtained for CTx and CTx + high MI CDUS treated tumors Figure A-D.Furthermore, we did not observe any significant differences in mean track length, where a reduction would indicate a vascular breakdown Figure A, E-G. FIn mice with 4T1 tumors, conventional ultrasound analysis showed a slight reduction in PE after CTx + high MI CDUS treatment of 4T1 tumors Figure .2; mouse: 127 (110-160) µm2; p < 0.0001; Figure Next, a histological comparison of human and mouse tumor tissue was performed to understand the stronger vascular response of the murine TNBC to high MI CDUS. We found that human TNBC lesions contain more connective tissue, which is often localized around the tumor microvessels. In contrast, murine 4T1 tumors contain only a very low amount of connective tissue, and microvessels are in closer contact with tumor cells Figure A & B. ThFor the first time, we prospectively investigated whether a diagnostic high MI CDUS protocol can alter tumor perfusion to improve the therapy outcome of neoadjuvant chemotherapy in breast cancer. With our diagnostic high MI CDUS protocol, we did not detect changes in the vascular morphology and function of breast cancer in patients. However, the application of this protocol in murine TNBC resulted in a significantly reduced tumor rBV, reduced microbubble velocity, and a lower amount of perfused blood vessels. Furthermore, we found differences in tumor morphology between the two species, possibly explaining the divergent effects of high MI CDUS.® microbubbles to investigate the effects of destructive CDUS on breast cancer vascularization during neoadjuvant chemotherapy.Clinically, diagnostic CEUS commonly uses nonlinear acoustic responses of microbubbles that already occur at acoustic pressures below 200 kPa Multiple preclinical studies In experiments focusing on improved drug delivery by sonopermeation, growth inhibition of murine prostate and pancreatic tumors The alternative approach to induce a vascular breakdown to trap chemotherapeutics was successfully demonstrated in murine breast ®, which has a very broad size distribution, with 80% of the microbubbles ranging between 3 - 9 µm in diameter ®, this leads to a wide range of frequencies (approx. 1-4 MHz) that can be used to induce nonlinear oscillation, which is favorable for clinical diagnostic applications at low acoustic pressure , mULM was used to identify discrete changes in the vascular morphology and function. However, also this sensitive post-processing technique Despite the lack of morphological and functional changes in the tumor vascularization, a slight but non-significant delay in tumor size reduction was observed in the high MI CDUS group during the first chemotherapy cycles. Although this may well be the result of undersampling, we decided to stop patient recruitment as it was clear that the intended positive effects on tumor perfusion were negligible. It is worth mentioning that we did not expect to observe significant improvements in the therapeutic outcome as the chemotherapy treatment regime, e.g., for TNBC with 4 cycles of epirubicin + cyclophosphamide every 2-3 weeks followed by 12 weekly cycles of paclitaxel combined with carboplatin is known to cause pCR in ~ 50% of patients The observation towards a somewhat slower response of high MI CDUS treated tumors to CTx has made us want to better understand the biophysical effects of our high MI CDUS protocol on tumor tissue and, therefore, we performed experiments on mice with TNBC using the same settings.Interestingly, opposed to the results obtained in patients, post-processing with mULM revealed a significantly reduced rBV and microbubble velocity directly after high MI CDUS in mouse TNBC. Histological analyses confirmed a significant reduction in the number of perfused tumor blood vessels. Despite the reduced perfusion after high MI CDUS, the amount and distribution of carboplatin were comparable to untreated tumors. Possible explanations for this are that carboplatin accumulated in the tumor before the vascular breakdown occurred, as the carboplatin infusion was initiated shortly before applying high MI CDUS. Additionally, carboplatin was mostly detected close to larger and more mature vessels, which might be less affected by oscillating or collapsing microbubbles. This hypothesis is also supported by other studies in hepatocellular carcinoma The question arises why we did not observe a vascular breakdown in human tumors. Although we used comparable ultrasound protocols to treat murine and human tumors, some differences between the experimental settings between mice and humans need to be mentioned as limitations of this study. First, murine tumors  are located closer to the body surface, resulting in less tissue between tumor and transducer and thus higher energy deposition. The attenuation coefficient in breast fat tissue is 0.73 dB/cm/MHz In summary, we here present a prospective clinical evaluation of high MI CDUS treatment for enhancing tumor vascularization to improve neoadjuvant chemotherapy in breast cancer. Regarding the various ultrasound settings that can be adjusted , we show that the current destructive ultrasound setting  is not suitable to improve tumor perfusion or response to neoadjuvant chemotherapy in patients, the latter even tending to be prolonged. We are aware that patient numbers are low but it is unlikely that a larger cohort of patients would have elucidated a clinically meaningful benefit of the ultrasound intervention. This is particularly true when taking our preclinical findings into account, where the high MI CDUS setting even leads to a partial vascular disruption. This stronger response of the preclinical than the clinical tumors may be explained by differences in their vascular and stromal composition. Only the combination of preclinical and clinical examinations in the present study enabled the identification of the tumors' vascular and stromal characteristics as an important factor for a successful application of microbubble assisted ultrasound treatment. Thus, there seem to be a small range in which high MI CDUS is either purely diagnostic, improves perfusion and vessel permeability, or induces vascular ablation. Intense research is still required to identify ideal ultrasound settings and microbubble formulations. In this regard, ultrasound protocols inducing a strong oscillation might be favorable to induce effects in highly desmoplastic tumors. Additionally, the effects may also strongly depend on the tumor tissue characteristics, which should be an integral part of future biophysical ultrasound examinations and enable the identification of suitable tumor entities and the preselection of responding patient subpopulations.Supplementary methods, figures and tables.Click here for additional data file.


As a highly prevalent disease among women worldwide, breast cancer remains in urgent need of further elucidation its molecular mechanisms to improve the patient outcomes. Identifying hub genes involved in the pathogenesis and progression of breast cancer can potentially help to unveil mechanism and also provide novel diagnostic and prognostic markers. In this study, we integrated multiple bioinformatic methods and RNA in situ detection technology to identify and validate hub genes. EZH2 was recognized as a key gene by PPI network analysis. CENPL, ISG20L2, LSM4, MRPL3 were identified as four novel hub genes through the WGCNA analysis and literate search. Among these, many studies on EZH2 in breast cancer have been reported, but no studies are related to the roles of CENPL, ISG20L2, MRPL3 and LSM4 in breast cancer. These four novel hub genes were up-regulated in tumor tissues and associated with cancer progression. The receiver operating characteristic analysis and Kaplan–Meier survival analysis indicated that these four hub genes are promising candidate genes that can serve as diagnostic and prognostic biomarkers for breast cancer. Moreover, these four newly identified hub genes as aberrant molecules in the maintenance of breast cancer development, their exact functional mechanisms deserve further in-depth study. Although current breast cancer therapeutic methods have been well developed and improved, latest data showed that breast cancer still has a high mortality rate among women worldwide. Thus, there is still an urgent need to explore the potential molecular mechanisms for improving the patient outcomes3. Among a wide range of different bioinformatics tools, weighted gene co-expression network analysis (WGCNA) algorithm is the most commonly used method for gene co-expression network research. By constructing co-expression gene modules and associating external information, the key gene modules and potential hub genes can be identified6. In general, hub genes show high connectivity in the gene co-expression network, which often located in the upstream of the gene regulatory network and play a predominant role in the gene network coordination8. Therefore, identification of potential novel hub genes is of great significance for exploring the mechanism of tumor initiation and progression. Selection of appropriate dataset is an important prerequisite for screening hub genes, and multiple types of datasets that were generated from different platforms are now available in the public database9. To explore the best potential of these datasets, it would be of great advantage to integrate them for downstream analysis. To achieve this goal, we used Robust Rank Aggregation (RRA) analysis algorithm for the process of breast cancer datasets. RRA is a reliable bioinformatic method that can remove substantial inter-study variations and statistical analysis difficulties existed in individual studies via integrating the gene expression profiles of different cross-platform datasets10. It has been used in various malignant tumor studies, such as in hepatocellular carcinoma, colorectal cancer, lung cancer and thyroid cancer14. Heterogeneity is one of the characteristics of tumor cells, which is reflected by different expression patterns of genes at the transcription level15. Analyzing and identifying the temporal and spatial heterogeneity information of RNA expression can be of great value to reveal the structural relationship between tissues and cells, as well as to uncover the potential functions of genes in disease state. RNA in situ detection technology can be used for studying the heterogeneity of RNA expression, and under the condition of maintaining tissue and cell morphology integrity, it can obtain the spatial localization and abundance of intracellular RNA at the single cell level16.In the past decade, applications of high-throughput chip and sequencing technologies have resulted in accumulation of a wealth of novel research data resource that can be analyzed by a series of bioinformatic methods, providing a novel approach to explore the molecular mechanism of tumorigenesis and tumor developmentIn this study, we first integrated DEGs from multiple breast cancer datasets based on RRA algorithms, and then identify the key gene of known functions using PPI network analysis. At the same time, WGCNA algorithm was applied to construct a weighted gene co-expression network and screen for potential novel hub genes related to breast cancer. The diagnostic performance and prognostic value of these novel hub genes were evaluated and their possible molecular mechanisms in breast cancer were explored by bioinformatic methods. Finally, we also made full use of RNA in situ detection technology to detect the expression abundance and spatial localization of each hub gene at single cell level, and further analyzing the expression differences and correlations, such that to validate those results from the bioinformatic analysis mentioned above.p value < 0.05 as screening criteria to obtain the robust DEGs between breast cancer tissues and normal tissues. A total of 512 robust DEGs were identified, containing 202 up-regulated genes and 310 down-regulated genes (Supplementary Table S1). Supplementary Fig. S1 shows the top 20 most significant up-regulated and down-regulated robust DEGs obtained by RRA methods from these eight different datasets. Of those, COL11A1  and S100P  were the two most significant up-regulated genes. Meanwhile, LEP  and FGF2  were the two most significant down-regulated gene in breast cancer tissues.Differentially expressed genes (DEGs) of eight datasets from the GEO database were integrated to perform RRA analysis, and the characteristics for each dataset are shown in Table To gain insight into the known biological processes and pathways involved in breast cancer, GO functional enrichment analysis and KEGG pathways analysis of 512 robust DEGs were performed. The results showed that those robust DEGs were significantly enriched in 720 GO terms and 9 KEGG pathways, respectively (Supplementary Table S2). Figure The PPI network of the 512 DEGs, including 493 nodes and 2993 edges was constructed via STRING database (minimum required interaction score: 0.4). By ranking the PPI network nodes using 9 topological analysis methods including both local- and global-based algorithms from cytoHubba plugin of Cytoscape software, we found the EZH2 score ranked in the top 10 by 9 algorithms  derived from the above RRA analysis to find key gene modules in breast cancer. After series of quality assessment for gene expression matrix, we set soft threshold as 5  to construct and validate the scale-free network  and brown module  were most correlated to breast cancer  were significantly higher in the subjects aged ≤ 51 years, and high expression of CENPL, ISG20L2, MRPL3, and LSM4 were associated with lymph node metastasis and higher SBR grade (P < 0.05)   were also significantly higher in breast cancer tissues than normal tissues . Immunohistochemistry analysis from The Human Protein Atlas database also showed that these four hub genes were up-regulated in protein expression level in breast lobular carcinoma . To elucidate the underlying mechanisms of abnormal up-regulation of these four hub genes in breast cancer, we first investigated the association between gene expression and their methylation levels. DiseaseMeth version 2.0 analysis displayed that the mean methylation levels of CENPL, MRPL3 and LSM4 were all significantly reduced in breast cancer compared to normal breast tissues , meaning that the four hub genes all possess good diagnostic performance.To explore the diagnostic performance of each hub gene in breast cancer, we first performed ROC analysis to assess the diagnostic performances of the four hub genes for detecting breast cancer using TCGA_BRCA dataset, and their AUC values  were showed in Fig. To estimate the prognostic value of each hub gene in BC patients, we conducted overall survival (OS) analysis using Kaplan–Meier survival method based on three different datasets that contains several thousand breast cancer samples. The Kaplan–Meier curves of each hub gene showed that the difference between high expression groups and low expression groups were significant . And the higher expression levels of these four hub genes were significantly associated with the poor OS of breast cancer patients .We measured the expression abundance and spatial localization of the five hub genes by RNA in situ detection technology. The results indicate that the expression of these five hub genes were mainly distributed in the cytoplasm and nucleus, and the amount of signal originates from hybridization to each probe varied greatly. Among these five genes, ISG20L2 showed the highest expression level, while CENPL and LSM4 have fewer signal Fig. a–d. CompAs a highly prevalent tumor disease worldwide, the complex mechanism involved in the development of breast cancer has not been fully elucidated so far. Thus, identifying potential hub genes involved in breast cancer is not only helpful to elucidating the molecular mechanisms, but also possessing great potentials for the searching of an effective diagnostic biomarkers and prognosis predictors. Previously, hub genes were mainly produced by using a small-scale dataset in most research and showed distressing inconsistent results. In this paper, we applied 10 breast cancer datasets to identify and validate potential novel hub genes so as to guarantee the credibility of the results.19. Meanwhile, the KEGG pathway enrichment analysis showed that 22 genes from those robust DEGs were most associated with PI3K—AKT signaling pathway, which serves as a pivotal intracellular signaling path that plays a crucial role in cell cycle regulation and thus involved in breast cancer development20. In addition, we also found that those robust DGEs were significantly enriched in PPAR signaling pathway, EC—receptor interaction, AMPK signaling pathway, and multiple studies have shown these pathways activation are participated in the development and progression of breast cancer and affect the final outcomes23. Based on the PPI networks analysis of robust DEGs, EZH2 was found to be a key gene in the development of breast cancer, and this has been shown in various breast cancer-related studies25.First, 512 robust DEGs between breast tumor tissues and normal breast tissues were identified using RRA method. Functional annotation analysis revealed that the robust DEGs significantly enriched in GO terms were associated with proliferation and energy metabolism, such as extracellular matrix organization, extracellular structure organization, mitotic nuclear division, regulation of lipid metabolic process, and glycosaminoglycan binding, which are implicated in the progression of tumor cell26. Interferon stimulated exonuclease gene 20 Like 2 (ISG20L2) encodes a 3'–5' exoribonuclease that involved in the 12S pre-rRNA processing, as a target gene of miR-139-3p, has been reported to take part in the pathogenesis of hepatocellular carcinoma28. Mitochondrial ribosomal protein L3 (MRPL3), which belongs to the L3P ribosomal protein family, encodes a 39S subunit protein, and plays a regulatory role in the process of Combined Oxidative Phosphorylation29. Small nuclear ribonucleoprotein Sm-like4 (LSM4) encodes a member of the LSM family of RNA-binding proteins, has an important role in pre-mRNA splicing by mediating U4/U6 snRNP formation, and this gene has been reported involved in the pathogenesis of pancreatic cancer31. Interestingly, Joseph S. Baxter et al32 have also identified that LSM4 is one of 110 target genes at 33 breast cancer risk loci based on Capture Hi-C technology, which supports the conclusions of the present study. In this study, our data not only showed the expression level of CENPL, ISG20L2, LSM4 and MRPL3 were strongly associated with age, lymph node metastasis and higher SBR grade, but also significantly upregulated in triple negative breast cancer patients compared to non-triple negative breast cancer. Furthermore, they are also correlated with the expression of the tumor cell proliferative marker MKI67 (Ki-67), indicating that these four genes may play vital roles in the pathogenesis and progression of breast cancer.Subsequently, a total of 104 hub genes associated with breast cancer were found using the “WGCNA” approach. Among those 104 hub genes, we chose four significantly up-regulated genes, including CENPL, ISG20L2, MRPL3, and LSM4 as hub genes of interest due to the reason that they have rarely been studied in breast cancer and closely related to clinicopathological variables of BC patients. Centromere Protein L (CENPL) is a component of the CENPA-CAD  complex, which participated in the assembly process of kinetochore proteins, mitotic progression and chromosome segregationNext, ROC analysis revealed that the mRNA expression levels of these four hub genes had excellent diagnostic performance for breast cancer. Prognosis analysis showed that these hub genes were high risk genes, and the higher expression levels were related to poorer prognosis for breast cancer patients. Early diagnosis and accurate evaluation of prognosis play an important role in improving the prognosis of breast cancer patients. thus, these four hub genes also have potentials to serve as promising candidate diagnostic biomarkers and prognosis predictors for breast cancer.33. Moreover, in cBioPortal database, we found that the abnormal expression of the above hub genes in breast cancer were associated with genetic alterations. In Summary, we think that the gene regulation complexity and up-regulation mechanism of the four hub genes should be further studied.Alternatively, we have also undertaken a preliminary analysis of up-regulation mechanism of these four hub genes refer to DiseaseMeth 2.0 and cBioPortal Database. In DiseaseMeth 2.0 database, the correlation analysis of DNA methylation patterns of hub genes with mRNA expression revealed that CENPL, MRPL3 and LSM4 were hypomethylated in breast cancer samples compared with adjacent normal ones, while ISG20L2 was hypermethylated in breast cancer sample. Generally speaking, the DNA methylation patterns negatively correlates with mRNA expression, which is consistent with the observed up-regulation of CENPL, MRPL3 and LSM4 in breast cancer, while the latest research suggested DNA hypermethylation can lead to mRNA upregulation34. Given that the specific functions of those novel hub genes remain unclear, additional research are required to investigate the underlying molecular mechanisms in breast cancer.In addition, we used GSEA to further explore the biological functions of the four hub genes in breast cancer and the results showed that the high-expression groups of CENPL, ISG20L2, MRPL3 and LSM4, were significantly enriched in pathways related to cell proliferation such as “cell cycle” pathway. The results of GSVA were in accordance with the GSEA results. Cell-cycle dysregulation is one of the hallmarks of cancer and several researches have reported that cell cycle disturbance is the most important mechanism for cancer occurrence and progression36, but no studies are related to the roles of CENPL, ISG20L2, MRPL3 and LSM4 in breast cancer. Thus, these four novel hub genes as aberrant molecules in the maintenance of breast cancer progression, their exact functional mechanisms deserve further in-depth study.Finally, RNA in situ detection technology was applied to detect the five hub genes obtained based on bioinformatics methods, and the results showed that the expression of those five hub genes are different but the spatial localization are similar. While verifying the expression differences of each gene, the correlation analysis showed that the CENPL, ISG20L2, MRPL3 and LSM4 was correlated with EZH2 expression. Since EZH2 are currently more studied in breast cancer, and the four novel hub genes  and EZH2 are involved in similar signaling pathways derive from GSEA results, we speculate that each novel hub gene has correlation with EZH2 expression, which were verified by the RNA in situ detection results. As an oncogene, growing evidence has identified EZH2 was closely related to breast cancer development and progression through multiple molecular mechanismshttp://www.ncbi.nlm.nih.gov/geo/), and processed using R package “GEOquery”37. The RNA sequencing data normalized by FPKM method, which contains 1066 breast cancer samples and 112 adjacent normal breast tissues samples, were downloaded from The Cancer Genome Atlas (TCGA) data portal  . At the same time, survival time and vital status of each breast cancer sample in TCGA_BRCA dataset were also extracted and used for subsequent overall survival (OS) analysis. The mRNA expression data and clinicopathological characteristics of 1,904 breast cancer samples in METABRIC dataset were acquired from the cBioPortal website , of which the mRNA expression levels were determined by Illumina Human v3 microarray and normalized by logarithm38. In addition, the raw data of three breast cancer related datasets  derived from the same microarray platform (GPL570 Affymetrix human genome U133A U133 Plus 2.0 array) were collected separately, then merged and preliminarily cleaned using the “GEOquery” package. The SVA function and Combat function were used to standard and remove the batch effect of three different datasets40. The merged dataset (GEO_BRCA dataset) was used to validate the diagnostic performance of single hub genes in breast cancer. Furthermore, Breast cancer samples microarray gene expression data from three datasets  and concomitant follow-up information were also acquired from the Gene Expression Omnibus (GEO) database using “GEOquiry” package and merged with TCGA_BRCA dataset using the “ComBat” function, which served as TCGA_GEO BRCA dataset (including 1596 breast cancer patients) to explore the prognostic value of novel hub genes.Thirteen different datasets comprising eleven datasets from GEO database, one dataset from TCGA database (TCGA_BRCA dataset) and one METABRIC dataset, thousands of breast cancer samples and 280 normal breast tissues samples, were included in our study. The eight series matrix files and corresponding platform annotation information files in RRA analyses were downloaded from Gene Expression Omnibus (GEO) database  in each dataset were sorted by their fold change value. subsequently, R package “RobustRankAggreg” 10was applied to integrate the ranked DEGs of 8 datasets from GEO database so that to find the most important and robust DEGs. Finally, those robust DEGs were determined according to the thresholds: |log2fold change|≥ 1 and P < 0.05.To discern the DEGs between breast cancer and normal breast tissue in each dataset from GEO database. The “limma” package in R was adopted to normalize the gene expression data and conduct differential gene expression analysis42. The GO terms or KEGG pathways with Adjusted P values less than 0.01 indicated statistical significance. Plus, bubble plots were used for visualizing the top 20 enrichment results of GO terms and KEGG pathways.To identify the biological functions and pathways of those robust DEGs, Gene Ontology (GO) Function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the “clusterProfiler” R packagehttps://string-db.org/, database version 11.0) to construct a PPI network43. Nine topological algorithms in Plug-in CytoHubba44, consisting of “MCC”, “MNC”, “Degree”, “BottleNeck”, “EcCentricity”, “Closeness”, “Radiality”, “Betweenness” and “Stress” were selected to identify the hub genes in PPI, and the top 10 genes in each topological algorithm were viewed as most stable key gene in PPI analysis. Moreover, the plug-in Molecular Complex Detection (MCODE) 45in Cytoscape software was also applied to analyze and recognize the modules in the PPI network. All parameters of the above analysis procedure used were set at default values.To identify the key gene of known functions in breast cancer, 512 robust DEGs were mapped to STRING database  was extracted from TCGA breast cancer dataset and associated with sample information to construct a sample clustering tree. Second, appropriate soft threshold value  was selected to convert the correlation matrix into adjacency matrix. Subsequently, the resulting adjacency matrix was further converted to topological overlap matrix (TOM) by the TOM similarity algorithm. Referring to the TOM‐based dissimilarity calculation formula, these 3776 genes were classified into different gene modules marked by different colors. Third, the minimal module size was set as 50 genes and the height cut-off as 0.25 to merge the highly similar gene modules. Meanwhile, the correlation value between each module's module eigengene (ME) and samples information were calculated using Pearson correlation coefficient. The candidate gene modules were identified based on the degree of correlation between the module's ME values and samples traits. Genes with gene significance (GS) value greater than 0.3 and module membership (MM) value greater than 0.6 in candidate modules were defined as hub genes for breast cancer. These genes may have stronger association with the progression and development of breast cancer. Finally, these hub genes were further filtered out based on bioinformatics analyses and literature searches.To screen potential novel hub genes related to breast cancer, the WGCNA algorithmhttp://bcgenex.ico.unicancer.fr) 47is a widely used statistical mining online tool for exploring the "correlation", "expression" and "prognostic" analyses of genes of interest in breast cancer by incorporating published annotated breast cancer transcriptomic data (DNA microarrays [n = 11 359] and RNA-seq [n = 4 712]). In this part of the study, the association of each hub genes expression with clinicopathological features in breast cancer was assessed using the "expression" analysis functionalities in bc-GenExMiner v4.6, and the clinicopathological parameters used in this study mainly contained nodal status (N), Age status, Pathological tumor stage, Scarff Bloom & Richardson grade status (SBR), Ki67 status and Basal-like (PAM50) & triple-negative breast cancer status, etc. A p value less than 0.05 was considered statistically significant.Bc-GenExMiner v4.6 (http://gepia.cancer-pku.cn/) database and The Human Protein Atlas  database were used to validate the differential expression of each hub gene between breast cancer tissue and normal breast tissue from gene expression and protein levels separately.The Gene Expression Profiling Interactive Analysis  curves analysis was used to evaluate the diagnostic value of each hub gene using in TCGA_BRCA dataset and GEO_BRCA dataset respectively.With the aid of R package “pROC”P < 0.05 was considered as difference significant. In parallel, hazard ratio (HR) value at 95% confidence interval (95% CI) of each hub gene was also calculated. HR greater than 1 suggested that the gene increase the risk of breast cancer, and HR less than 1 indicated that the gene was a beneficial factor for breast cancer. Moreover, the prognostic value of each novel hub gene was further assessed using the "prognostic" analysis functionalities in bc-GenExMiner v4.6 Platform.To assess the prognostic value of each hub gene, both the samples in METABRIC dataset and TCGA_GEO_BRCA dataset were divided into high-expression group and low-expression group based on each hub gene’s best separation cut-off values. Using built-in “survminer” package and “survival” package in R software, the overall survival (OS) rates were calculated via the Kaplan–Meier method, and the difference in the OS rates between high expression group and low expression group of each hub gene was compared by the log-rank test, http://bioinfo.hrbmu.edu.cn/disease meth/) is a database that integrates massive methylation data from microarray and sequencing results, providing the methylation status annotation information of human diseases49. This web database was used to compare the difference of methylation levels of each hub gene between breast cancer and normal breast tissues.The human disease methylation database  was used to investigate the correlation of genetic alteration and gene expression in breast cancer.The genetic alteration data for each hub gene in the METABRIC dataset samples at the cBioPortal website . Cutoff value of differential KEGG pathways was set |logFC|˃0.2, and P < 0.01 was regarded as statistically significant.51 and the main steps include: Design of padlock probe complementary to the target RNA, after the padlock probe hybridize to its target and the padlock probe is connected into a ring through specific Splint R DNA ligase, the rolling-circle amplification (RCA) is initiated under the action of primer and Phi29 DNA polymerase, and Finally, fluorescently labeled probes were added to achieve signal detection. For high detection efficiency, three padlock probes were designed for each hub gene in this study . The cell lines used in this study include: MCF10A, MCF7, MD-MB-231 and SKBR3. Image analysis and quantification of signal intensity from each probe was performed in CellProfiler software. A minimum of 1000 cells was counted for each cell line probe set, and wilcox—test was conducted to compare the rolling circle products (RCPs) of each hub genes between human breast epithelial cell line (MCF10A) and breast cancer cell lines . The expression correlation of these hub genes in breast cancer cell lines were analyzed with pearson method. P-value less than 0.05 was considered statistically significant.RNA in situ detection technology was used to determine each hub gene expression at the cellular level. Specific operations were performed referring to literature reported by Ruijie Deng et alNot applicable.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.Supplementary Information 12.Supplementary Information 13.Supplementary Information 14.


Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer with an estimated 750,000 cases diagnosed annually in the United States. Most cases are successfully treated with a simple excision procedure, but ~5% of cases metastasize and have a 5-year survival rate of 25-45%. Thus, identification of biomarkers correlated to cSCC progression may be useful in the early identification of high-risk cSCC and in the development of new therapeutic strategies. This work investigates the role of complement factor H (CFH) in the development of cSCC. CFH is a regulatory component of the complement cascade which affects cell mediated immune responses and increases in complement proteins are associated with poor outcomes in multiple cancer types. We provide evidence that sun exposure may increase levels of CFH, suggesting an immunomodulatory role for CFH early in the development of cSCC. We then document increased levels of CFH in cSCC samples, compared to adjacent normal tissue (ANT) routinely excised in a dermatology clinic which, in paired samples, received the same level of sun exposure. We also provide evidence that levels of CFH are even greater in more advanced cases of cSCC. To provide a potential link between CFH and immune modulation, we assessed immune system function by measuring interferon gamma (IFN-γ) and FOXP3 in patient samples. IFN-γ levels were unchanged in cSCC relative to ANT which is consistent with an ineffective cell-mediated immune response. FOXP3 was used to assess prevalence of regulatory T cells within the tissues, indicating either a derailed or inhibitory immune response. Our data suggest that FOXP3 levels are higher in cSCC than in ANT. Our current working model is that increased CFH downstream of sun exposure is an early event in the development of cSCC as it interferes with proper immune surveillance and decreases the effectiveness of the immune response, and creates a more immunosuppressive environment, thus promoting cSCC progression. Cutaneous squamous cell carcinoma (cSCC) is typically treated by tumor excision with a success rate of >95%. As a minority of cSCC are known to metastasize and cause clinically serious disease, research on cSCC is sparse and therapies for the ~5% of cases that do metastasize are limited, resulting in a 5-year survival rate of only 25-45% , 2. HoweEvidence is accumulating that the tumor microenvironment is a key factor in the progression of all tumor types. The immunomodulatory nature of the tumor microenvironment has been shown to be particularly relevant due to discovery of the clinical efficacy of treatments targeting immune checkpoints. In this work, we focus on the potential role of two complement regulatory proteins, complement factor H (CFH) and complement factor I (CFI), in the immune response to tumors.As regulatory proteins, CFH and CFI modulate the complement cascade at multiple points, but their most impactful effect is through reducing levels of several potent anaphylatoxins (including C3a and C5a). Local anaphylatoxin production increases recruitment of both innate and adaptive immune cells to the tumor. In addition, recently characterized as immune checkpoints , C3aR anin vivo  to Affiliated Laboratories BioRepository (ALBR). Additionally, the Midwestern Institutional Review Board approved the use of these clinic-based biorepository samples at Midwestern University (AZ#807). The single criterion for the collection of tissues for these procedures is a biopsy-proven diagnosis of cSCC. The initial diagnosis and classification of cSCC type was completed at the clinic as part of routine patient care prior to transfer of the sample to the research laboratory. No exclusion criteria were outlined in the original IRB protocol but samples from patients with a known blood-borne communicable disease were not used. All tissue specimens were obtained from patients who consented to donate excised tissue removed during Mohs surgery. For viable tissue used in explant cultures, cSCC tissue from the center of the apical side of the tumor was removed before processing the sample for histology. If needed for wound closure, the surgeon removed adjacent normal tissue (ANT) and these were matched with the tumor sample for paired analysis.2 incubator. When approximately 80% confluent, cultures of mixed cultures were passaged onto glass coverslips for immunofluorescence.Tissues were processed for culture and immunofluorescence as described in Belden et al. . BrieflyFor immunofluorescence, explant cells were grown on eight-well chamber slides, washed with 1x PBS, fixed for 15 minutes with 4% paraformaldehyde in 1x PBS, rinsed with 1x PBS, and incubated in 0.05% Triton X-100 in 1x PBS for 5 minutes to permeabilize the cells, and followed by blocking with 1% BSA in 1x PBS for one hour. Blocking reagent was aspirated and cells were rinsed with 1x PBS and incubated overnight with 1:200 mouse anti-CFH  at room temperature. Primary antibody was omitted as a negative control. After washing with 1x PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 568 phalloidin (1:500) for one hour at room temperature, washed with 1x PBS, mounted in fluoromount with DAPI (Electron Microscopy Sciences) and imaged with a Zeiss Apotome microscope.® TGX™ Precast Protein gel (Bio-Rad), transferred to a low fluorescent PVDF membrane, and blocked using 5% NFDM  for one hour at room temperature. Primary antibodies in 1% NFDM used were 1:200 rabbit monoclonal histone H3 antibody (D1H2) , 1:1000 rabbit anti-GAPDH , 1:200 mouse monoclonal CFH (OX-24) , 1:200 mouse monoclonal IFN-γ (Santa Cruz Biotechnology), and 1:200 mouse monoclonal antibody FOXP3 (F9) (Santa Cruz Biotechnology). 1:10,000 AlexaFluor® 790 (Abcam) or 1:5,000 HRP-conjugated (Santa Cruz Biotechnology) were used as secondary antibodies. All blots were performed in triplicate and relative protein expression was measured using either an Odyssey® CLx (LI-COR Biotechnology) or ChemiDoc XRS (BioRad) imaging system. Band intensities were normalized to either GAPDH or H3 using Image J software (NIH).Total protein from patient derived frozen tissue samples (Affiliated Dermatology Laboratory) were isolated using RIPA buffer , DNaseI and DTT following an established protocol . Forty mSlides of formalin-fixed, paraffin-embedded (FFPE) cSCC and ANT tissue sections were either purchased (US Biomax & Biochain) or obtained from ALBR. A standard immunohistochemistry protocol was performed by baking the sections at 60°C for 60 minutes, de-paraffinizing by placing in xylene followed by reducing concentrations of ethanol (100% to 70%). De-paraffined sections were permeabilized using 0.25% Trypsin with no EDTA. For heat induced epitope retrieval, FFPE tissue sections were incubated in either citrate buffer (CFH) or basic buffer (FOXP3) at 95°C for 25 minutes, followed by blocking and overnight incubations at 37°C, and 4°C with primary antibodies, mouse anti-CFH (OX-24)  and rabbit monoclonal anti-FOXP3 (Cell Marque). CFH slides were incubated with AP-conjugated secondary and permanent red stain. FOXP3 slides were incubated with an HRP-conjugated secondary and DAB stain (for array slides) or an AP-conjugated secondary antibody and permanent red stain (for ALBR slides). Mayer’s Hematoxylin was used as a counterstain (nuclei) followed by mounting with Flourmount G mounting medium. Slides were imaged using bright field Olympus (DP73 camera) microscope at 40X, 100X and 400X magnifications. An isotype control was performed for each tissue type and against each antibody species. Semi-quantitative analysis of IHC images was performed based on colorimetric intensity over a specified area of tissue sections using a 0-3+ scale, with 0 indicating no staining, 1+ indicating <10% staining, 2+ indicating 10-50% staining, and 3+ indicating >50% staining, and verified using ImageJ (NIH) .Analysis of GTEx (Genotype-Tissue Expression) data was performed using non-parametric tests in GraphPad Prism v9. A Mann-Whitney two-tailed T-test was used to analyse unpaired data and a two-tailed Wilcoxon matched-pair signed-rank test was used to analyse paired data. Statistical significance did not vary with or without removal of outliers using the ROUT method of identifying outliers. Correlation of paired data was performed using Spearman’s rank-order correlation coefficient.post-hoc power analysis was applied to data that did not show significance. Data analysis was performed with GraphPad Prism. Alpha (α) was set at 0.05 for all statistical tests and data with a p ≤ 0.05 were considered statistically significant.Band intensities from immunoblot data were normalized to histone H3 or GAPDH. A ROUT test with an alpha value of 0.05 was used to identify potential outliers within each dataset. D’Agostino and Shapiro-Wilk tests followed by a T-test (paired or unpaired depending on the comparison) were used for determining the significance of differences for normally distributed independent variables. D’Agostino and Shapiro-Wilk tests followed by a Mann–Whitney U-test was used for determining the significance of differences between two non-normally distributed independent variables. A To assess the effect of sun exposure on CFH and CFI, we interrogated all available data in the GTEx portal. In the dataset of 473 exposed (lower leg) and 387 non-exposed (suprapubic) unpaired patient samples, CFH mRNA expression is higher in exposed skin than non-exposed skin (p < 0.0001) but no difference in CFI mRNA levels was seen.To determine the effects of sun exposure using paired patient samples, we analysed the subset of 278 subjects with GTEx values for both sun-exposed and non-exposed skin. Analysis of these paired data suggests that CFH mRNA in exposed skin increased significantly over non-exposed skin (p < 0.0001)  from sun exposed skin were collected during routine Mohs micrographic surgery as previously described and primary cultures were established for both cSCC and ANT samples . PatientAnalysis of band intensities indicates a 1.76- and 1.53-fold increase in expression for CFH and FHL-1 respectively when compared to paired ANT  (Levels of interferon gamma (IFN-γ) in the patient-derived cSCC samples above was quantitated next. This pro-inflammatory cytokine may mark an effective immune response and synthesis of CFH in keratinocytes has been reported to be under the control of IFN-γ –7. ImmunThe transcription factor FOXP3 is often used to detect the presence of regulatory T cells which are reflective of a dampened immune response. Immunoblotting revealed a band at the expected molecular weight for FOXP3 . This allows us to directly assess levels of these proteins in newly diagnosed cSCC excised in a dermatology clinic as part of routine practice (ALBR Samples), which may not be as progressed, with the more advanced cSCC samples utilized in most studies. In addition, as the samples in these analyses retain the tissue integrity, they allowed for determination of colocalization of these proteins within the tumor tissues.As shown in Next, levels of FOXP3 in ANT and cSCC tissues from routine clinic , the link between CFH is extended to newly diagnosed and non-invasive cSCC. Although the observed CFH elevation was small, this is perhaps due to the relatively small size of the tumor and the amount of normal tissue included in the patient samples excised in the Mohs procedure. Indeed, we suspect that this may be the reason that CFI was not reliably detected in our samples as others have found that CFI levels in tissues are lower than CFH and consistent with GTEx data Figure 1As our collective data sets provide support for sun exposure affecting overall levels of CFH, we suggest that immune modulation is an early event in the development of cSCC. This finding is not unexpected as an increase in CFH is expected to reduce both innate and adaptive immune responses to tumors, a necessary step in tumor progression. Furthermore, the fact that IFN-γ levels do not increase in our cSCC samples when compared to ANT may be consistent with an ineffective immune response. It is interesting that we do not see elevated levels of IFN-γ as this cytokine has been shown to increase CFH secretion , 7. It iThis work documents both an increase in CFH and FOXP3 in cSCC but does not directly address the relationship between these two findings. Published work suggests a plausible causal link between increased CFH secreted from cSCC and immunoevasion as suggested by increased FOXP3 levels. Expression of CFH by keratinocytes and cSCC cells lines has been well documented , 7 and iAlthough not immune cells, growing evidence suggests that cancer cells express anaphylatoxin receptors and are able to respond to increased anaphylatoxin levels. Specifically, a wide variety of cancers and cancer cell lines express C3aR and C5aR and respond by increased motility and activation of the ERK1/2 pathway to promote growth –37. MostData presented here helps to solidify the relationship between CFH and tumorigenesis but they also raise many questions about the role of CFH in cancer progression. Specifically, in addition to its role in cSCC, a role for CFH has been described for hepatocellular and clear cell renal cell carcinomas (ccRCC) but not does not appear to promote squamous cell lung carcinoma , 38. In There are many remaining questions regarding the role of CFH in cSCC and clarification of these points may have direct impact on treatment of patients with cSCC. To clarify whether the increase in CFH contributes to or is a result of tumor development, it will be important to establish signaling through anaphylatoxin receptors in cSCC and solidify the evidence of immune modulation. The ability of IFN-γ to increase CFH secretion by keratinocytes documented in cell lines can be replicated in patient samples needs to be investigated. This latter point is of particular importance as a current therapy, imiquimod (used in the treatment actinic keratosis and some cSCC), is associated with enhancing IFN-γ production to mount an effective immune response  through CFH may be elevated in cSCC tumors excised from patients seen in a routine Mohs microsurgery. This elevation in CFH appears to be independent of sun exposure and may act through derailing an effective immune response. Immune checkpoint therapies targeting anaphylatoxin receptors may be an effective treatment for cSCC in the future.https://gtexportal.org/home/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The Genotype-Tissue Expression (GTEx) Project portal at The studies involving human participants were reviewed and approved by Western IRB (WIRB Protocol #20142461) to Affiliated Laboratories BioRepository. The patients/participants provided their written informed consent to participate in this study. Approval was also obtained from Midwestern University IRB (AZ#807).EJ, CU, and AP conducted experiments and contributed to data analysis. SE, RA, KL, and EH analysed data and edited the manuscript. KL and EH conceived of the project and coordinated the research endeavours. All authors have approved the manuscript.This research was funded by MWU Research Facilitation Grants to KL and EH and by graduate student funds to EJ.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.