N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM  These cellular amalgamations can be useful in promoting stem cell differentiation down various lineages [Organoids, microtissues or tumour-spheroids are terms used to describe small clusters of cells cultured lineages ,2, inclulineages –3, inclulineages ,5. Theselineages . This pain vitro, their incremental complexity over traditional 2D monolayers may create greater challenges for data reproducibility, potentially contributing to the so-called reproducibility crisis [et al. conducted an economic analysis where they estimated that irreproducible preclinical research costs the USA $28 billion annually [While 3D tumour-spheroids may offer better tools to interrogate biology y crisis . The repy crisis ,8. Freedannually . Tumour-STEMCELL Technologies already sells cryopreserved mouse intestinal, hepatic or pancreatic organoids . STEMCELMany different strategies are used to assemble cells into organoids, microtissues or tumour-spheroids, including non-adherent culture surfaces , microflN-Acetyl-l-cysteine (NAC), a ROS scavenger [To evaluate the above approach, we mass-manufactured uniform tumour-spheroids from C4-2B prostate cancer or MCF-7 breast cancer cells using in-house fabricated microwells. We optimised a freeze–thaw protocol using CryoStor CS10 and mitigated reactive oxygen species (ROS)-induced damage in thawed tumour-spheroid cultures by supplementing the medium with cavenger . The tum. 2. 2.12 and 37°C. Cultures were passaged at approximately 80% confluency using 0.25% Trypsin/EDTA (v/v) (Thermo Fisher). Cells were re-seeded at 1500 cells/cm2 in fresh flasks and expanded as required for tumour-spheroid manufacture or further experimentation.The prostate cancer cell line, C4-2B  and brea. 2.22. A wad punch (Amazon.com) was used to punch 1.6 cm diameter discs from the PDMS sheets. Using silicone glue, disc inserts were anchored in 24-well plates. Each disc insert had approximately 1200 microwells and was thus capable of generating approximately 1200 tumour-spheroids. To sterilise plates, 1 ml of 80% (v/v) ethanol was added to the well, and the plate was centrifuged at 3000×g to displace bubbles from the microwells and under the inserts. Following centrifugation, the plates were submerged entirely in an 80% ethanol bath for 1 h at room temperature. The microwells were washed thrice with sterile deionised water (Thermo Fisher Scientific), dried overnight at 60°C in an oven, and stored at room temperature until needed. Prior to cell seeding, microwell surfaces were treated with sterile 5% Pluronic F-127 Sigma-Aldrich) in Dulbecco's phosphate buffered saline (DPBS) (w/v). Pluronic solution (1 ml) was added to each well, and the plate was centrifuged at 3000×g for 10 min to displace air bubbles from microwells. Pluronic F-127 absorbs and coats the PDMS surface, thereby reducing protein adsorption and cell attachment onto the PDMS surface, which promotes cell aggregation [Tumour-spheroids were assembled in microwells, using microwell plates fabricated in-house, as previously described ,26. Our regation . Excess . 2.35 cells, 2.4 × 105 cells or 4.8 × 105 cells in 1 ml of HG-DMEM supplemented with 10% FBS and 100 U/ml Pen/Strep. Cells were pelleted into microwells by centrifuging the plate at 300×g for 5 min. The distribution of cells into the microwells and their subsequent aggregation was confirmed using an Olympus CKX14 microscope, digital camera  and imaging software . Prior to cryopreservation, tumour-spheroids were cultured for 48 h in a 5% CO2 atmosphere at 37°C.As previously described , C4-2B o. 2.42 and 37°C incubator for 24 h. Following this 24-h revival culture, the Happy Cell ASM suspension was inactivated using the Inactivation Solution that is provided with the product. This process results in the tumour-spheroids no longer being buoyant in the Happy Cell ASM, sinking to the bottom of vessel and allows for full media exchange. The tumour-spheroids were washed with HG-DMEM and then approximately 50 tumour-spheroids were resuspended in 200 µl of fresh HG-DMEM supplemented with Happy Cell ASM, 10% FBS and Pen/Strep in each well of ultra-low binding 96-well plates (Corning 3474 Costar). Images of the tumour-spheroids were captured using an Olympus CKX14 microscope. Tumour-speroid diameters were measured using a digital camera  and imaging software  before suspending into Happy Cell ASM media. These data were compared with the non-cryopreserved tumour-spheroids cultured over the same time period.The C4-2B and MCF-7 tumour-spheroids were gently and aseptically aspirated from microwells. Approximately 1000 tumour-spheroids per millilitre of cryoprotectant were loaded into 1.2 ml internal threaded polypropylene Cryo.s freezing tubes . We assessed C4-2B tumour-spheroid cryopreservation in standard laboratory cryopreservation solution, 90% FBS supplemented with 10% DMSO, or alternatively in the commercial cryoprotectant Cryostor CS10. The manufacturer's instructions were followed when using CS10. Briefly, the culture media was removed from wells and the tumour-spheroids were resuspended in cold (4°C) CS10 solution. The suspended tumour-spheroids were incubated at 4°C for 10 min. The CS10 or FBS plus DMSO suspended tumour-spheroids were transferred to a controlled cooling rate container  and frozen at −80°C for 24 h. The tumour-spheroid-containing vials were transferred to liquid nitrogen for extended storage periods. The cryopreserved tumour-spheroids were rapidly thawed at 37°C in a water bath, gently swirling the submerged cryotube base. The cryomedia was diluted 1 : 10 with HG-DMEM supplemented with 1% PenStrep, 10% FBS and 10 µM NAC. After observing that C4-2B or MCF-7 tumour-spheroids amalgamated in the HG-DMEM culture media, compromising downstream cultures, we subsequently cultured tumour-spheroids in medium supplemented with Happy Cell ASM medium  and 10% FBS and Pen/Strep. Tumour-spheroids are semi buoyant in Happy Cell ASM medium, and this prevents their aggregation. In the optimised protocol, tumour-spheroids were cryopreserved in CS10, then revived in 1 ml of medium formulated from HG-DMEM, supplemented with Happy Cell ASM, 10% FBS and Pen/Strep and 10 µm NAC. Revival cultures were performed in 5 ml flat base screw cap tubes , leaving the screw cap loose in a humidified 5% CO. 2.52, 37°C incubator. AlamarBlue fluorescence was recorded at 544 nm excitation and 590 nm emission from the bottom of the plate using a CLARIOstar Plus microplate reader (BMG LABTECH) every hour for 4 h. A titration of C4-2B or MCF-7 cells was characterised in parallel, providing a viable cell standard curve for the AlamarBlue assays.The metabolic activity of post-thawed tumour-spheroids was assessed using AlamarBlue solution containing Resazurin (Thermo Fisher Scientific). Resazurin is a blue-violet dye that is reduced by NADH or NADPH reductase enzymes, turning pink or into Resorufin, which has a strong fluorescence emission ,30. The . 2.6The viability of tumour-spheroids was assessed using LIVE/DEAD cell viability solution (Thermo Fisher Scientific), following manufacturer's instructions. Post-thaw recovered or drug-treated tumour-spheroids were washed with PBS following the AlamarBlue assay. As a negative control, tumour-spheroids were devitalised in 80% ethanol for 30 min, yielding a dead cell control. Approximately 10 tumour-spheroids were distributed into wells in a black 96-well plate and stained with 2 µM calcein-AM  and 4 µM of ethidium homodimer-1 . The tumour-spheroids immersed in the staining solution were incubated in the dark for 45 min at room temperature. Tumour-spheroids were washed with PBS to remove excess stain and then assessed using an Olympus FV3000 confocal microscope.. 2.7The DNA content of tumour-spheroids was quantified as secondary means to estimate viability. The DNA quantification was performed using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) as per the manufacturer's instructions. Briefly, tumour-spheroids were lysed using a combination of 1% Tween 20 in TE buffer  and two freeze/thaw cycles, as described previously . Twenty-. 2.82 incubator at 37°C following drug treatment. After 72 h, the culture media was supplemented with 3% AlamarBlue to determine the metabolic output of the different cultures in response to the drug. Fluorescence was read at 544 nm excitation and 590 nm emission using a CLARIOstar Plus microplate reader (BMG LABTECH), 4 h following the addition of AlamarBlue. Parallel drug assays were performed with C4-2B or MCF-7 cells cultured in monolayers in standard tissue culture-treated 96-well plates (Corning Costar). Each well was seeded with 5000 cells in 200 µl HG-DMEM supplemented with 10% FBS and 100 U/ml pen/step. Cultures were maintained in a 5% CO2 and 37°C incubator for 24 h before initiating Docetaxel treatment. Like tumour-spheroids, the monolayer cells were incubated with the drug for 72 h before the addition of AlamarBlue to the medium. During the metabolic AlamarBlue assays, a titration of the cancer cells was performed in parallel to function as a standard curve. The quantity of DNA in both the drug-treated cryopreserved and non-cryopreserved tumour-spheroids, as well as in the monolayer cultures, was assessed at the end of the metabolic assays to validate any gain or loss in cell number.The drug response of cryopreserved tumour-spheroids was performed using a cytotoxic drug, Docetaxel . After p. 2.9n = 4). Graph generation and statistical analysis were performed using GraphPad Prism version 8 software. The significance of the difference between the groups was calculated by one-way analysis of variance (ANOVA) following Tukey's post-hoc test. A p-value less than 0.05 was considered statistically significant.Data are presented as mean ± s.d. for four biological replicates . While the size and shape of the tumour-spheroids was not perfectly uniform, the microwell platform offers relatively good control over these features and high efficiency.In preliminary experiments, we aimed to establish an ideal tumour-spheroid size and cryopreservation medium for process optimisation. Tumour-spheroids were formed from 100, 200 or 400 C4-2B or MCF-7, and then cultured for 48 h and characterised using light microscopy. As depicted in figure 1b). Relative cell viability was greatest for 100 cell tumour-spheroids cryopreserved in Cryostor CS10, and the Cryostor CS10 cryopreservation medium is advantageous in this application as tumour-spheroids are slightly buoyant in this medium which results in reduced settling and tumour-spheroid aggregation. In subsequent studies, tumour-spheroids were assembled from 100 cells each and cryopreserved in Cryostor CS10. The diameter of tumour-spheroids generated from 100 C4-2B or 100 MCF-7 cells was assessed before and after cryopreservation. C4-2B tumour-spheroids (123.9 ± 14.3 µm) had a significantly greater diameter than MCF-7 tumour-spheroids (99.9 ± 20.0 µm), but in both cases diameters were similar before and after cryopreservation .Next, we compared cryopreservation outcomes for tumour-spheroids generated from 100, 200 or 400 C4-2B cells preserved in either 90% FBS + 10% DMSO or in Cryostor CS10. Following thaw, tumour-spheroids were assessed using Thermo Fisher's LIVE/DEAD cell viability kit b. Relati. 3.2a). We assessed the viability of tumour-spheroids cultured in HG-DMEM or HG-DMEM supplemented with Happy Cell ASM, and found that neutral buoyancy resulted in greater cell viability (figure 2b). We speculate that when tumour-spheroids aggregate that this can make the cells more vulnerable to shear stresses induced during culture manipulation and that, for this reason, there is a viability benefit realised when culturing in neutral buoyancy medium.While tumour-spheroids of 100 cells each cryopreserved in CS10 yielded high cell viability, the tumour-spheroids had a propensity to aggregate with each other in downstream cultures. Tumour-spheroid aggregation is problematic, as this uncontrolled aggregation process yields variably sized tumour-spheroids, which have different surface area to volume ratios and establish different diffusion gradients. These characteristics can influence cell biology as well as how tumour-spheroids respond in drug assays –34. Addifigure 2c,d shows that the conversion of AlamarBlue fluorescence was greater for non-cryopreserved C4-2B and MCF-7 tumour-spheroids, respectively. Tumour-spheroids cultured in Happy Cell ASM media outperformed HG-DMEM controls, but remained metabolically less active than non-cryopreserved controls.To better quantify how the addition of Happy Cell ASM to the culture system might influence cell viability, we assessed tumour-spheroid metabolic activity of fresh or thawed tumour-spheroids cultured in HG-DMEM supplemented with 10% FBS plus 1% Pen/Strep or HG-DMEM supplemented with Happy cell ASM, 10% FBS and 1% Pen/Strep. Figure 2. 3.3Previous literature suggests that NAC protects mitochondria from damage following cryopreservation . To assea) or MCF-7 (figure 3b) tumour-spheroids were permitted to recover in medium supplemented with 10 µM NAC, their metabolic activity was similar to non-cryopreserved tumour-spheroids. The DNA content in fresh and cryopreserved tumour-spheroids was measured using the PicoGreen assay. The C4-2B cryopreserved tumour-spheroids recovered in either HG-DMEM or Happy Cell ASM media had a similar quantity of DNA content compared to non-cryopreserved tumour-spheroids (figure 3c). The DNA content in cryopreserved MCF-7 tumour-spheroids was slightly reduced in Happy Cell ASM (72.2 ± 1.2 µg) and HG-DMEM (70.0 ± 4.2 µg) compared to non-cryopreserved Happy Cell ASM (75.6 ± 1.5 µg) and HG-DMEM (73.1 ± 3.2 µg) cultured tumour-spheroids, respectively (figure 3d). These differences were not statistically significant. NAC treatment during the thaw and recovery period did not alter the DNA content of the tumour-spheroids (figure 3e). These data suggest that cell number is not changing in tumour-spheroids during the cryopreservation process, and that manipulation with NAC is modifying respiration rather than tumour-spheroid cell content. The DNA content of C4-2B cryopreserved and non-cryopreserved tumour-spheroids were significantly greater than MCF-7 tumour-spheroids (figure 3e).Using this optimised recovery medium formulation, we assessed tumour-spheroid metabolic activity using the AlamarBlue assay as well as tumour-spheroid DNA content with or without cryopreservation . When C4figure 3. 3.4The optimised manufacturing, cryopreservation, revitalisation protocol was used in the drug screening assays; see schematic in n = 3) to confirm the observations. The viability images of the drug-treated cryopreserved and freeze–thawed tumour-spheroids from the other biological replicate experiments are presented in electronic supplementary material, figures S5 and S6.Cryopreserved and non-cryopreserved tumour-spheroids appeared similar in Live/Dead assays . Few deaa) or MCF-7 (c) tumour-spheroids showed almost identical responses to Docetaxel treatments when evaluated using the AlamarBlue assay. The AlamarBlue signal declined significantly in C4-2B and MCF-7 monolayer cultures, evident from the lowest concentrations of Docetaxel . The difference between monolayer and cryopreserved or non-cryopreserved tumour-spheroid cultures was significant from 1 to 100 nM of Docetaxel . C4-2B cells, cultured in monolayers, were almost metabolically inactive with Docetaxel greater than 5 nM (a). MCF-7 cells showed the lowest metabolic activity at 100 nM (c).Cryopreserved and non-cryopreserved C4-2B a or MCF-or MCF-7 c tumour-ocetaxel a,c. The ocetaxel a,c. C4-2han 5 nM a. MCF-7 t 100 nM c.Figureb,d). The effect of Docetaxel was dose-dependent for both tumour-spheroids and monolayer cells. The dose-response curves were also similar for cryopreserved and non-cryopreserved tumour-spheroids. DNA content of C4-2B monolayer cells sharply declined from 0.1 to 5 nM and remained steady up to 100 nM (b). The declination of DNA content was gradual with the increment of Docetaxel concentration for MCF-7 monolayer cells (d). The percentage of DNA content for both C4-2B and MCF-7 tumour-spheroids was significantly greater than corresponding monolayer controls across the same drug concentrations . The experiments were conducted in three biological triplicates (n = 3). Results from biological replicate experiments are presented in electronic supplementary material, figures S7 and S8. These data were consistent with our previous report, which also studied these cell populations [When DNA was quantified in these cultures, trends similar to the metabolic assays were observed b,d. The o 100 nM b. The deer cells d. The petrations b,d. The ulations .. 4Not long ago, some laboratories generated many of their own reagents, including sophisticated reagents such as antibodies or Taq polymerase. Today, most reagents are available as standardised and quality-controlled products. Deferring reagent development to supply companies provides a more effective division of labour, ultimately reduces costs, contributes to reagent consistency, and has significantly accelerated the rate of discovery in the biomedical sciences. Tumour-spheroids are growing in popularity as a culture tool, and it may be possible for tumour-spheroids, microtissues or organoids to evolve to become standardised reagents for some applications. There is already precedent for this. We cite STEMCELL Technology's sale of cryopreserved intestinal organoids ,12, but C4-2B and MCF-7 cells were assembled into tumour-spheroids using a microwell platform, after which they were cultured for 48 h. Microwell platforms enable efficient mass production of uniformly sized tumour-spheroids –17,37–40Following initial cryopreservation steps, we thawed tumour-spheroids, and cultured them in HG-DMEM supplemented with 10% FBS. Visual observation of these cultures identified several problems; first, while microwell culture enabled the assembly of uniform tumour-spheroids, their subsequent culture in standard well plates led to uncontrolled and undesirable tumour-spheroids clumping, or amalgamation. Second, the clumping or disruption of clumps appeared to damage cells on the surface of tumour-spheroids, leading to death. Third, some settling and clumping of tumour-spheroids appeared likely to occur during cryopreservation in 90% FBS + 10% DMSO, but tumour-spheroids appeared to settle less  in CS10 cryopreservation medium.We took the view that it would be ideal if the tumour-spheroids could be maintained in a suspension, thereby minimising clumping, and maintaining the useful discrete uniform tumour-spheroid product generated in the microwell platform. Based on these observations, we used CS10 cryopreservation medium in subsequent experimentation to minimise tumour-spheroid aggregation during the cryopreservation process. Happy Cell ASM, is a novel neutral buoyancy medium, that, according to the manufacturer, is a ‘unique, low viscosity, low density, cell culture reagent that permanently suspends cells'. Based on these promising properties, we trialled thawing tumour-spheroids into and culturing tumour-spheroids in Happy Cell ASM medium. These two modifications contributed to better maintenance of discrete tumour-spheroids with high viability.2O2 and ·OH, as well as contributing to glutathione replenishment and protecting sulfhydryl groups on the mitochondrial membrane [In addition to tumour-spheroid assessment with LIVE/DEAD viability dye, we characterised tumour-spheroid metabolism using the AlamarBlue assay. Despite high viability, tumour-spheroid metabolic activity was significantly reduced following cryopreservation. Previous studies have reported impaired cellular aerobic respiration and mitochondrial function following cryopreservation –44. A simembrane . We postIn our study, thawed tumour-spheroids were exposed to NAC for 24 h in a ‘revitalisation' step prior to their use in the drug assays. NAC was depleted by washing the tumour-spheroids before transferring them into the drug assay plate. Thus, our study did not evaluate how the combination of NAC and drug might have influenced the drug assay outcomes. Data in electronic supplementary material, figures S2 and S3 show the significant increase in metabolic activity in non-frozen tumour-spheroids exposed to 10 µM NAC. Noting the caveat that we have not tested this, we presume that having NAC in the culture medium used in the drug assay may confound assays outcomes; possibly elevating respiratory activity in cells that are dying and where you might expect to observe a declining respiratory signal. For this reason, it is necessary to have a staged process where thawed tumour-spheroids are treated with NAC, the NAC then eliminated, and then the tumour-spheroids used in drug assays. Critically, it does appear that cell respiration in tumour-spheroids is compromised following cryopreservation. A more refined process would be one that reduces the impact of cryopreservation, perhaps allowing the tumour-spheroids to be used directly, or earlier following thaw, in drug assays. Agilent Seahorse XF Technology allows precise quantification of cell metabolic activity, and a rational next step might be to characterise metabolic activity prior to and following cryopreservation, and to use the Agilent Seahorse XF to assess the benefits of various cryopreservation medium supplements to either protect against damage or to more rapidly restore metabolic function. Likely, it will be possible to shorten or eliminate the ‘revitalisation’ step currently integrated into our protocol.The cryopreserved tumour-spheroids were assessed for drug response using the anti-cancer drug, Docetaxel. Docetaxel binds and stabilises microtubules, leading to cell cycle arrest and apoptosis . Treatme. 5Herein, we optimised a process for manufacturing cancer tumour-spheroids in microwells, their cryopreservation in CS10 medium, recovery in Happy Cell ASM, supplemented with 10% FBS and 10 µM NAC, then distribution of tumour-spheroids into 96-well plates again in Happy Cell ASM, supplemented with 10% FBS to study drug response. The success of this process revolves around three critical considerations: (i) Optimisation is facilitated by using a uniform tumour-spheroid product, and we recommend a microwell platform. (ii) Tumour-spheroid aggregation should be minimised during freezing, manipulation and during culture. This can be achieved in part through buoyancy conferred by CS10 cryopreservation medium and by Happy Cell ASM cell culture medium. (iii) Cell respiration is impaired following cryopreservation, but this can be rescued by including a recovery culture where the medium is supplemented with NAC. Limitations to the described process include the necessity of a recovery step, and the fact that we have not yet adapted the process to more challenging primary cell populations. Nevertheless, this process does provide an initial road map that may guide future efforts to develop tumour-spheroid banks, that contain tumour-spheroids ready-to-go as standardised inputs into experiments conducted around the world.


For patients eligible to undergo breast-conserving surgery (BCS) after neoadjuvant chemotherapy, accurate preoperative localisation of tumours is vital to ensure adequate tumour resection that can reduce recurrence probability effectively. For this reason, we have developed a 3D-printed personalised breast surgery guide (BSG) assisted with supine magnetic resonance imaging (MRI) and image 3D reconstruction technology, capable of mapping the tumour area identified on MRI onto the breast directly using dual positioning based on the manubrium and nipple. In addition, the BSG allows the colour dye to be injected into the breast to mark the tumour region to be removed, yielding more accurate intraoperative resection and satisfactory cosmetic outcomes. The device has been applied to 14 patients from January 2018 to July 2023, with two positive margins revealed by the intraoperative biopsy. This study showed that the BSG-based method could facilitate precise tumour resection of BCS by accurately localising tumour extent and margin, promoting the clinical efficacy in patients with breast cancer as well as simplifying the surgical process. Breast cancer has surpassed lung cancer as the most diagnosed cancer worldwide (approximately 2.3 million in 2020), accounting for 24.5% of all cancer cases . For earOf many imaging modalities commonly employed to determine tumour extent and location, such as computed tomography, mammography, sonography and magnetic resonance imaging (MRI), MRI is considered more suitable for identifying residual tumours after NACT due to its higher accuracy ,7,8. A vThe emergence of three-dimensional (3D) printing has advanced the capability to fabricate 3D biomedical devices with architecturally complex features, such as biomimetic implant scaffolds ,17, micrThis study proposes a customisable 3D-printed surgical guide based on supine MRI and image 3D reconstruction technology for breast-conserving surgery after NACT, followed by surgical verification and postoperative pathological assessments. First, the eligibility of patients for BCS was determined with detailed patient characteristics. The 2D supine MRI image slices were then reconstructed to represent 3D breast organs, within which the 3D tumour can be segmented. Based on the digital information of segmentation and reconstruction, a surgical guide was designed with the ability to position the device on the surface of the breast and locate the internal tumour extent accurately. Finally, the clinical practice was investigated by applying the device to 14 patients, followed by postoperative pathological analysis.Patients who met the following criteria were admitted: (1) the patient was diagnosed with locally advanced invasive breast cancer confirmed by our hospital; (2) the patient completed NACT successfully and was willing to undergo BSC after NACT; (3) the shrinking pattern of the initial tumour after NACT was diagnosed to be concentric contraction . PatientDetailed patient information, such as age, contact information, operation method, initial tumour diameter, tumour size after neoadjuvant chemotherapy and operation time, was recorded. The tumour characteristics are summarised in To minimise the localisation error, the supine position identical to the operation’s was adopted during MRI image acquisition. Breast MRI images were acquired using a 3.0 T MRI system  with plain scan AxT1, AxT2, Ax + Sag C+ and injection of 15 mL contrast agent  at a rate of 2.5 mL/s, followed by rinsing with 10 mL of 0.9% normal saline see a–c. The Based on the supine MRI image series (DICOM format), tumour segmentation was carried out slice-by-slice by our experienced radiologists using a digital medical design platform , as indicated by the red contours in © ZRapid Tech, Suzhou, China), and a 0.1 mm thin plate was added as the supporting structure. The model was then sliced with a layer thickness of 0.1 mm. Prior to printing, the resin was stirred to ensure it was well mixed and subsequently left for 30 min to remove the bubbles. Following the recommendation of the manufacturer, the light intensity and layer thickness were chosen to be 375 mW cm−2 and 0.1 mm, respectively, ensuring that the curing depth was slightly larger than the layer thickness necessary to enhance the bonding of two neighbouring layers while achieving the desired balance between printing speed and vertical resolution. The entire printing was conducted at a constant ambient temperature of 25 °C. After printing, the patterned BSG was immersed in isopropyl alcohol for 15 min to soften the supporting structure for removal. The part was then cleaned using alcohol and stored in a sterile cabinet after drying for further use.The STL files containing the surface geometric information of the designed BSG were fed into a 3D slicer software for slicing essential for 3D printing. The device was then fabricated using a stereolithography (SLA) 3D printer that creates 3D parts by crosslinking photocurable materials in a layer-wise fashion. Each device was attached with a barcode containing the patient’s personal information see , thus avAfter general anaesthesia and sterilisation, the patient’s upper limb on the surgical side was outstretched. The surgical guide plate was placed gently on the breast, where the nipple was inserted into the target hole of the surgical guide to ensure the correct position. Then, the azimuth was adjusted by rotating the device to overlap the patient’s manubrium with that of the surgical guide plate such that the breast and the plate were completely fitted and the projection of the lesion was well overlapped a. The meThe specimens were frozen at −20 °C in a freezer for frozen biopsy pathology analysis. According to the surgeon’s marking points, the material was collected using the method of radial sections perpendicular to the margin. After staining, the samples were placed under the microscope to examine whether there were tumour cells in the cutting margin and to measure the distance between the resection margin and the tumour edge.Postoperative pathological assessment was conducted to clarify the tumour characteristics such as type, grade and stage. Immunohistochemistry and tumour molecular pathology tests were performed, including oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) immunohistochemical staining and Ki-67 proliferation index tests.From January 2018 to July 2023, 20 patients with locally advanced breast cancer were enrolled in this study, and 6 patients refused to use a BSG for BCS following a discussion. Breast cancer is the most common malignant tumour globally . NACT haPostoperative care is also indispensable. The appearance and dryness of the incision were monitored, and the dressing was changed regularly to prevent infection. The drainage tube was removed by examining the amount and colour of the daily drainage fluid. The pain assessment was conducted for the patient. Psychologists were assigned to patients to take special care of patients’ mental health. The patients were also provided with appropriate dietary advice.The consistency of the tumour information (location and margin) identified by imaging tools preoperatively and those applied on the patient’s breast intraoperatively is vital for the localisation accuracy of the BSG-based approach. The entire process of this strategy can be divided into three stages: (1) image acquisition; (2) digital tumour information transferred from images to the device; (3) tumour information projection from the device to the patient’s breast. Each step is affected by several factors. During the first stage, the imaging tool selection is crucial for accurately identifying the tumours. MRI has been reported to be the superior imaging modality for assessing the morphology and extent of breast cancer and is widely used for surgical decisions to achieve better negative surgical margins ,8,29. HoRegarding the limitations of this study, the clinical application cases may be insufficient, as the BSG was applied to 14 patients from January 2018 to July 2023. More operable patients will be enrolled continuously in the future to provide statistical meaning to this study. In addition, tumour segmentation based on MRI data was conducted manually slice-by-slice for each patient, which is not only time-consuming but also lacks consistency. Artificial-intelligence-based segmentation technology might be an optimal alternative to automate this procedure .Aiming at facilitating the clinical treatment for patients eligible for BCS after NACT, a BSG-based tumour localisation method is proposed in this study and was applied to 14 patients from January 2018 to July 2023. The patient-specific BSG was developed based on supine MRI data, assisted with image segmentation, image 3D reconstruction and 3D-printing technologies, making it possible to project the tumour location and margin identified from MRI onto the patient’s breast precisely, thereby promoting the accuracy of resection and reducing the re-excision rate. The BSG employs a dual-positioning method to improve the positioning accuracy when applied to the breast, while the injection of blue dye offers the surgeon visual guidance for precise tumour resection. Twelve negative resection margins were achieved, accounting for ~85.7%. The excised tumours exhibit a regular hemispherical-like shape, indicating satisfactory cosmetic outcomes. Compared with traditional localisation methods, the BSG-based strategy is less invasive, radiation exposure-free, simpler and possesses a shorter operation time. In addition, it mitigates the dependency on the skills and experience of the surgical team, improving the surgical reliability and procedural standardisation that are important to clinical practice. In the future, more prospective trials are still needed to verify the safety and efficacy of this technology in depth.


We introduce a new spatial statistic, the weighted pair correlation function (wPCF). The wPCF extends the existing pair correlation function (PCF) and cross-PCF to describe spatial relationships between points marked with combinations of discrete and continuous labels. We validate its use through application to a new agent-based model (ABM) which simulates interactions between macrophages and tumour cells. These interactions are influenced by the spatial positions of the cells and by macrophage phenotype, a continuous variable that ranges from anti-tumour to pro-tumour. By varying model parameters that regulate macrophage phenotype, we show that the ABM exhibits behaviours which resemble the ‘three Es of cancer immunoediting’: Equilibrium, Escape, and Elimination.We use the wPCF to analyse synthetic images generated by the ABM. We show that the wPCF generates a ‘human readable’ statistical summary of where macrophages with different phenotypes are located relative to both blood vessels and tumour cells. We also define a distinct ‘PCF signature’ that characterises each of the three Es of immunoediting, by combining wPCF measurements with the cross-PCF describing interactions between vessels and tumour cells. By applying dimension reduction techniques to this signature, we identify its key features and train a support vector machine classifier to distinguish between simulation outputs based on their PCF signature. This proof-of-concept study shows how multiple spatial statistics can be combined to analyse the complex spatial features that the ABM generates, and to partition them into interpretable groups.The intricate spatial features produced by the ABM are similar to those generated by state-of-the-art multiplex imaging techniques which distinguish the spatial distribution and intensity of multiple biomarkers in biological tissue regions. Applying methods such as the wPCF to multiplex imaging data would exploit the continuous variation in biomarker intensities and generate more detailed characterisation of the spatial and phenotypic heterogeneity in tissue samples. Multiplex images provide exquisitely detailed information about the spatial distribution and intensity of up to 40 biomarkers within two-dimensional tissue regions, creating challenges and opportunities for quantitative analysis. Although stain intensities are measured on a continuous scale, they are typically converted into discrete labels to simplify subsequent spatial analysis. In this paper we propose a new spatial statistic, the weighted pair correlation function (wPCF), which exploits, rather than neglects, the continuous variation in stain intensity contained in multiplex images, and can characterise both spatial and phenotypic heterogeneity.As proof-of-principle, we apply the wPCF to synthetic data that resemble multiplex images of solid tumours. We generate data from an agent-based model (ABM) that simulates macrophage-tumour interactions. The wPCF shows how the continuous label describing macrophage phenotype is spatially related to categorical labels associated with tumour cells and blood vessels. We demonstrate that correlation functions can categorise spatial relationships in a manner which is interpretable and quantitative.The methods we present can be used to analyse both ABM simulations and multiplex imaging data, with applications that go beyond macrophage phenotype to include other biological processes that exhibit continuous variation . The immune landscape within solid tumours is complex and varied , 2, withribution –9. For esurvival .1 or M2, individual macrophages may exhibit a variety of behaviours. Further, their overall behaviour, or phenotype, may change over time in response to multiple microenvironmental cues [While macrophages are often classified as either Mtal cues . Macrophtal cues . It is dtal cues , 12–14.n = 35 patients (data reproduced from [In ced from ). Variat1 and M2 classes, or the seven subtypes shown in r between point pairs and quantify the correlation between marks at distance r. For example, the mark correlation function kmm determines whether the marks of two points separated by a distance r are spatially correlated: kmm(r) > 1 indicates that the marks of points separated by r are larger than the average mark, while kmm(r) < 1 indicates that they are smaller than the average [r [r. The wPCF addresses this, by identifying spatial interactions between points of one type and those whose mark is within a range of target values.Stratification of macrophage populations into discrete classes  of tumour growth that accounts for tumour-macrophage interactions and dynamic changes in macrophage phenotype. ABMs are well-suited for generating labelled point pattern data, since each cell is represented by an agent whose behaviour is determined by subcellular variables  and drives them towards a pro-tumour phenotype. At the same time, CXCL12 produced by perivascular fibroblasts biases the movement of these M2 macrophages towards neighbouring blood vessels. As they migrate out of the tumour, the pro-tumour macrophages express epidermal growth factor (EGF), a tumour cell chemoattractant. In this way, M2 macrophages facilitate metastasis by guiding the tumour cells towards the vasculature [The off-lattice, force-based ABM that we develop is motivated by an experimental study by Arwert et al  which inculature , 34.1 and M2) [et al. [p, whose dynamics depend on local levels of TGF-β and determine macrophage behaviour.The hybrid ABM developed in this paper builds on existing differential equation models , 36 and  and M2) –43 and t and M2) –46. Efti and M2)  and El-K [et al.  have devWe vary ABM parameters, and show that it generates a range of spatial patterns and qualitative behaviours that resemble ‘The Three Es of cancer immunoediting’ . In partThe remainder of the paper is structured as follows. We first describe our ABM, emphasising those features which make it appropriate for generating synthetic imaging data. We introduce the wPCF and illustrate how it can be interpreted as a series of cross-PCFs which vary continuously with the label of interest, here macrophage phenotype. We then apply the wPCF to synthetic data generated from our ABM, and show that it provides a more detailed description of the relationship between macrophage phenotype and spatial location than cross-PCFs. We define a ‘PCF signature’, consisting of two wPCFs and a cross-PCF, which describes the spatial relationships between blood vessels, tumour cells, and macrophages of each phenotype. The signature can be interpreted as a high-dimensional vector, and we apply principal component analysis (PCA) to reduce its dimensionality. We demonstrate a proof-of-concept classification algorithm by using the first 100 principal components to train a simple classifier which distinguishes between simulation outputs that correspond to the three Es of immunoediting . Finally, we calculate PCF signatures for dynamic ABM data, and show that a single simulation may transition between tumour Equilibrium, Escape and Elimination at different timepoints.in vivo, and includes phenotype-dependent interactions between macrophages, blood vessels and tumour cells. We outline the ABM here, and refer the interested reader to We present a 2D, multiscale, off-lattice ABM which extends an existing model of macrophage infiltration into tumour spheroids by accounting for continuous and dynamic variation in macrophage phenotype , 50. Theω), CSF-1 (c), CXCL12 (ξ), TGF-β (g) and EGF (ϵ). In the 2D Cartesian geometry, blood vessels are represented by fixed points which do not compete for space with the cell populations and which act as distributed sources of oxygen [The ABM distinguishes four cell types: stromal cells, tumour cells, necrotic cells, and macrophages. Their dynamics are influenced by five diffusible species: oxygen . Necrotic cells occupy space for a finite time period during which their size decreases linearly to zero and they are then removed from the simulation. Blood vessels also act as entry points for macrophages, which infiltrate the tissue and alter their phenotype  at rates which depend on local levels of TGF-β , we assume that chemotactic forces depend linearly on phenotype. The chemotactic forces acting on macrophage i, at position xi with phenotype pi, are therefore:c and ∇ξ are evaluated at xi. The forces Fi in Rint then the macrophage will attempt to kill the tumour cell, with M1 macrophages more likely to kill a tumour cell than M2 macrophages. We define a probability of cell kill per hour, Pφ, which is a monotonic decreasing function of p. We suppose further that, after a macrophage has killed a tumour cell, it experiences a ‘cooldown’ period of tcool hours during which it cannot attempt tumour cell killing. Thus, we associate with macrophage i a subcellular timer tφ,i that is updated in real time and set to zero on tumour cell killing. We define Pφ,i as:i is sufficiently close to attack multiple tumour cells then one is selected at random for cell death. Killed tumour cells are labelled as ‘necrotic’ and decay in the same way as other necrotic cells.We assume that when a macrophage and a tumour cell are within the interaction radius ϵ, is produced by M2 macrophages and undergoes natural decay. It is also a potent chemoattractant for tumour cells. For simplicity, we assume that macrophage i produces EGF at a rate which is linearly proportional to its phenotype pi, with constant of proportionality κϵ. Denoting by Dϵ and λϵ the assumed constant diffusion coefficient and natural decay rate of EGF, we suppose that its evolution can be described by the following reaction diffusion equation:δ(x) = 1 when x = 0 and δ(x) = 0 elsewhere. In x.The diffusible cytokine EGF, i (which may be a cell or a blood vessel), whose centre is located at xi =  at time t. We associate with object i a categorical label qi ∈ {B, M, S, T, N} which indicates whether it is a blood vessel, macrophage, stromal cell, tumour cell or necrotic cell. Given a target label Q ∈ {B, M, S, T, N}, the binary target function Θ indicates whether the label qi matches Q:In order to compute spatial statistics, we introduce the following notation. Consider an object r. We define a sequence of annuli, of inner radius rk and outer radius rk + dr where r0 = 0 and dr > 0. We denote by rk that is centred at the point x. If this annulus lies wholly inside the domain then Ik(r), is defined as follows:The cross-PCF identifies spatial correlations between objects with categorical labels that are separated by a distance A. If, at time t, this region contains NB blood vessels and NT tumour cells, then the cross-PCF, gBT(r), is given by:r ∈ [rk, rk + dr) and N is the total number of objects in the simulation. For each blood vessel, the cross-PCF compares the density of tumour cells in the annulus that surrounds it to NT/A, the expected density in the annulus under complete spatial randomness (CSR). Thus, gBT(r) > 1 indicates clustering of tumour cells at distance r from blood vessels and gBT(r) < 1 indicates anti-correlation, or exclusion, of tumour cells at distance r from blood vessels. Cross-PCFs for other pairs of categorical variables are defined similarly. We note that the cross-PCF is not necessarily symmetric .We calculate the cross-PCF for blood vessels and tumour cells by considering a region of area Q, qi) in wp ≤ 1, which describes how pi differs from a target phenotype, P. Multiple functional forms could be used for the weighting function. The relationship between the wPCF and cross-PCF is explored in more detail in P = 0.2. Then, wp ≈ 1 for cells whose phenotype pi is close to the target P and wp = 0 for those with |P − pi| > ΔP. We note further that wp → Θ as ΔP → 0. The choice of ΔP, therefore, represents a balance between obtaining a smoothly varying wPCF (reduced noise) and ensuring that the wPCF displays the signal of interest . Different values of ΔP are considered in We calculate the wPCF by replacing Θ with wp in P and blood vessels B, at lengthscale r, as follows:P across all macrophages (WP(P) replaces NT in wp = 0). Intuitively, the wPCF extends the cross-PCF by weighting the contribution of each macrophage based on how closely its phenotype matches the target phenotype.Replacing Θ and objects with a categorical label . In both examples, 200 crosses are uniformly distributed along the line y = 1, and 1000 circles are randomly placed throughout a square domain of edge length 2. In pi of a circle at  increases linearly with distance from the line y = 1 (pi = |1 − yi|); in pi = |1 − pi|2). The corresponding wPCFs are shown in the middle panels of P and distances r. For simplicity, when a wPCF is calculated over multiple values of the label p, we denote the resulting metric as wPCF. By construction, a circle at distance r from the nearest cross has label P ≈ r for (A) (and P ≈ r2 for (B)). The two wPCFs show strong clustering along these lines and exclusion at shorter distances for points with larger labels (above the dashed lines). The weaker clustering observed below the lines is explained as follows. Consider a cross at position . In (A), the largest label associated with a circle at distance r from this cross is p = r (if the circle is directly above the cross). Smaller labels can also be recorded, for circles at distance r which are offset from the cross in the y-direction. In the rightmost panels of wPCF for fixed values of the target label P. These curves can be interpreted as cross-PCFs for points whose labels pi are “close” to P, and show the strongest clustering at the expected values.In P1 and P2, say, in order to identify spatial correlations between objects with labels close to P1 and objects with labels close to P2 . We discuss such extensions in A natural extension to the wPCF considers objects with two continuous labels, t = 500 . These results show that varying c1/2 can generate a range of qualitative behaviours that mirror the three stages of cancer immunoediting [For a given set of parameter values, we run multiple realisations of the ABM and record simulation outputs at lues see . In Fig oediting . We summEquilibrium: a compact tumour mass, with macrophages confined to the surrounding stroma. The dominant macrophage phenotype is M1. Tumour growth is constrained, with tumour cells restricted to the mass and prevented from migrating to the vasculature by macrophage surveillance , the rate of macrophage extravasation is high, and some macrophages reach the tumour boundary through random exploration of the tissue. These macrophages kill tumour cells on contact, causing the tumour mass to decrease in size.Equilibrium (blue box) arises for low values of β causes the macrophages to transition to an M2 phenotype. The M2 macrophages migrate towards nearby blood vessels, up spatial gradients in CXCL12, and the CSF-1/EGF paracrine loop enables tumour cells to trail behind them. If these tumour cells reach the vasculature, we assume that tumour cells enter the vasculature and metastasise to other parts of the body. Therefore, we denote such simulations as tumour escape.Escape (orange box) occurs when macrophages migrate to the tumour so slowly that they do not overwhelm it. Further, exposure to TGF-c1/2). Tumour elimination occurs because the M1 macrophages are strongly attracted to the tumour mass and kill tumour cells before they are ‘reprogrammed’ to an M2 phenotype. Strong chemotactic sensitivity to CSF-1  occurs when the rate of macrophage extravasation is very high ). The wPCF identifies three distinct macrophage populations, rather than the two populations used in p ≈ 0 and blood vessels are not strongly correlated, as for the M1 population in p exhibit strong short range colocalisation with blood vessels, as for the M2 population in p ⪅ 0.6 which is strongly excluded from blood vessels at distances up to approximately 15 cell diameters. This distance corresponds to the approximate distance from blood vessels to the tumour core, suggesting that these macrophages are localised inside the tumour mass.In c1/2. These patterns resemble Equilibrium , wPCF and gBT(r), to characterise the pairwise spatial relationships between macrophages of different phenotypes, blood vessels, and tumour cells. We define the combination of these three statistics as a ‘PCF signature’ for our simulations.In p  There is a marked difference in the spatial localisation of macrophages with p ≈ 0 and those with p > 0.2, with mid-phenotype macrophages exhibiting short-range exclusion from blood vessels but not from tumour cells. In this case, macrophages which have not been exposed to TGF-β (p = 0) are restricted to the stroma while those with larger phenotype values cluster around the tumour mass, at distance from the blood vessels. The cross-PCF between blood vessels and tumour cells, gBT(r), indicates strong exclusion between blood vessels and tumour cells at distances of up to approximately 12.5 cell diameters, which is comparable to the exclusion distance between macrophages of intermediate phenotype and blood vessels.Equilibrium simulations  may not p = 1 cluster (r ≈ 0) with blood vessels and tumour cells. With the peak in gBT(r) near r = 0, this indicates the formation of perivascular niches containing tumour cells, blood vessels and M2 macrophages, as reported by Arwert et al. [gBT(r) for 2.5 ⪅ r ⪅ 12.5, this is characteristic of a central tumour mass populated with transitioning macrophages and the formation of perivascular niches.For the Escape simulations , macropht et al. . MacrophwPCF is extremely noisy (because there are very few tumour cells). Similarly, gBT(r) ≈ 1, since most simulations with this parameter set have no tumour cells. wPCF is similar to that shown in 2 macrophages cluster around blood vessels, and transitioning macrophages localise in the domain centre, at distance from the vasculature). This further suggests that the time courses for Elimination and Escape simulations may be similar at early times.Finally, for Elimination simulations , there awPCF, wPCF and gBT(r) to form the PCF signature described in the previous section. We then vectorised and concatenated the three spatial statistics, to form a high-dimensional vector . This process is described more fully in The parameter sweep described in wPCF, wPCF and gBT(r) capture sufficient information to distinguish between the three qualitative behaviours that the ABM exhibits .We then applied dimensionality reduction to the training dataset. In t = 500 could be used to classify it as Escape, Elimination or Equilibrium.The wPCFs and PCFs associated with the centroids of each cluster are presented in the inset in The 371 data points shown in c1/2) alongside randomly choosing four additional parameters (c1/2 and gcrit) in order to generate a wider potential variety of simulation outcomes  through to ‘Escape’  and, ultimately, to ‘Elimination’.The results presented in We calculate PCF signatures for this ABM simulation every 10 hours, and project them onto the first three principal components, using the process in t = 150). Once macrophages start to appear in the images, the trajectory moves away from the three clusters, due to noise in the wPCFs caused by the small number of macrophages relative to the t = 500 simulations which define the clusters (t = 250). As the number of macrophages increases, the trajectory moves closer to the Escape cluster . Finally, as the tumour cells are killed and removed from the simulation, the trajectory moves into the Elimination cluster .At early times, the images localise within the Equilibrium cluster . We showed that wPCFs and cross-PCFs can be combined to produce a high dimensional ‘PCF signature’ which characterises the relative locations of macrophages, tumour cells and blood vessels. Our results suggest that, given suitable training data, the PCF signature could be used to automate the classification of images or point patterns into different clusters, by using PCA to project the PCF signature onto a lower dimensional space.One advantage of testing the wPCF on synthetic data from an ABM is its ability to generate time-series data. Our analysis of dynamic output from an ABM simulation showed how the PCF signature may vary over time. A given simulation may transition between different states, suggesting that caution is needed when using a single time point to make predictions about a tumour’s future growth and response to treatment. This study also highlights one of the benefits of developing mathematical models of a biological system: the ABM can be used to investigate questions that would be challenging to address with existing experimental techniques. For example, it is not currently feasible to apply multiplex imaging to the same tissue at multiple timepoints.There are many directions for extending and improving the ABM. For example, including tumour cell intravasation would prevent situations in which an Escape signature can progress to Elimination at a later timepoint, as in The robustness of the wPCF should be tested through application to other types of ABMs that simulate tumour growth and generate similar outputs. In addition to the Chaste framework used here –53, otheIn this paper we used the wPCF and cross-PCF to describe and classify synthetic data generated by an ABM. However, the wPCF has application to a wider range of scientific fields. In particular, the wPCF can account for continuous variation in expression levels of cellular markers which characterise multiplex imaging modalities, such as multiplex immunohistochemistry or Imaging Mass Cytometry. Equally, the wPCF could be used to describe any structured cell population, for example continuous labels describing the stemness or differentiation status of cancer cells, the exhaustion level of T-cells, or the oxygen concentration experienced by cells within a tissue sample. Beyond biology, the wPCF could be used to analyse data from other applications in which PCFs have proven useful, including astronomy , 20 and Future work will involve applying the wPCF to multiplex imaging data, in order to validate its use in biological and clinical settings. Applying such statistics to medical images would enable their high-throughput, automated quantification and comparison in a manner that goes beyond expert visual inspection and is more interpretable than AI approaches , 66. We In this paper we presented a proof-of-concept SVM classifier to show how multiple statistics can be combined to classify data, with PCA acting as a dimension reduction technique which permits a classifier to be trained on high dimensional statistics without sacrificing their interpretability . This method has the potential to bridge the gap between classifiers applied to summary statistics, which are flexible and fast to train but generally do not include spatial data, and classifiers applied directly to images, which account for spatial features of images but are difficult to interpret, and require large training datasets and intensive computation. While the classifier described here works, there is considerable scope for optimisation in relation to i) the choice of statistics, ii) the choice of classification method, and iii) the choice of dimension reduction tool. In future work, we will explore alternative choices at each of these stages, in order to improve our pipeline for classification of point clouds based on their spatial structure. We will also use a wider range of techniques, such as topological data analysis, to characterise the outputs from different simulations and timepoints.This paper demonstrates an exciting proof-of-concept: statistics which describe different aspects of cell localisation can be combined to classify, describe and analyse synthetic and biological point clouds.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.S5 Appendix(PDF)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.S7 Appendix(PDF)Click here for additional data file.


Social media is increasingly being used among Infectious Disease (ID) programs. It can be a valuable recruitment tool in addition to providing education, self-promotion, and increasing collaborations across the ID community. We sought to analyze factors associated with increased engagement for our ID fellowship program’s Twitter account.We analyzed various metrics from consecutive Twitter posts and utilized the Twitter Analytics Dashboard to extract data from time of account inception. Impressions were defined as the number of times a tweet was seen by users, and engagements as a composite of interactions with a tweet. Tweet content was organized into educational, social, promotional, and other categories. Educational content was subcategorized into tweetorial, microbiology, question & answer (Q&A), and journal articles. A subset of tweets were analyzed for engagement from April 2022 to March 2023 for trends according to content and date the tweet was posted. Standard univariate descriptive statistical analyses, moods median test, and one-way analysis of variance was performed. Data analysis was performed using R version 3.4.0.460 tweets were posted from October 2020 to March 2023 . Educational subcategories showed a large amount of engagement related to microbiology, with specifically more retweets, likes, and profile clicks (Table 1). There was no difference in engagement based on time of day of tweet (Table 2), although profile clicks were significantly more likely if tweets occurred on a weekend (p=0.04). A subset of 284 tweets from April 2022 to March 2023 revealed that months where multiple content categories were tweeted resulted in the most engagement . Generally, more twitter posts correlated with more engagement with a Pearson correlation coefficient of 0.7.To increase engagement with social media, there should be frequent tweets with varied content categories. Subcategories of content such as microbiology tweets draw further interest and showcase a program's educational and clinical innovation.Indira Brar, MD, Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Gilead: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|ViiV: Advisor/Consultant|ViiV: Grant/Research Support|ViiV: Honoraria


A virus infection may lead the body to produce more immune cells of particular types or stimulate the production of new ones, both of which may have anti-leukemic effects. There has been no research on whether immune cells stimulated by varicella-zoster virus (VZV) infection have anti-leukemic effects. The objective of this investigation is to assess the impact of VZV infection on patients' long-term survival following allogeneic hematopoietic stem cell transplantation .This retrospective study investigated the association between varicella-zoster virus (VZV) reactivation and outcomes in 219 individuals who received allogeneic hematopoietic stem cell transplantation  at the Sun Yat-sen University’s First Affiliated Hospital. According to being diagnosed with VZV infection or not, these patients were grouped into two groups. The comparison of cumulative incidence of relapse, non-recurrent mortality, and overall survival (OS) was conducted between the two groups.Analyzing multivariate data, VZV reactivation was linked to lower relapse incidence in the group containing all individuals , patients suffering from acute myeloid leukaemia , and patients suffering from acute lymphoblastic leukaemia . Moreover, VZV reactivation was linked with decreased non-relapse mortality in all individuals , but no statistical significance was found for any disease subgroup. Further, VZV reactivation was an independent predictor for improved OS in the group containing all individuals , patients suffering from acute myeloid leukaemia , and patients suffering from acute lymphoblastic leukaemia .This is the first study to show that VZV reactivation following allo-HSCT is an independent predictor for lower relapse rates and improved OS, providing novel therapeutic approaches to improve patients’ long-term survival following allo-HSCT. Allogeneic hematopoietic stem cell transplantation  is an efficient therapy that contains the potential to cure hematologic malignancies. Although the incidence of post-transplant relapse is now being lowered by monitoring minimal residual disease , creatinRecent advances in tumour immunology and evasion mechanisms have rekindled interest in immunotherapies for cancer. Immune cells triggered by an infection may have antitumor effects, particularly in patients with viral infections. This is because natural killer cells and CD8+ T cells, specifically, cytotoxic T cell lymphocytes, are essential components of the immune response to viruses. These cells are also crucial effector cells in immunological reactions against cancer –9. In 20Despite the fact that VZV is a different type of herpes virus, unlike CMV reactivation, no research has been conducted on the association between VZV reactivation and the long-term prognosis of individuals following allo-HSCT. Therefore, the primary intent of this investigation is to assess the connection between VZV reactivation and the cumulative incidence of relapse (CIR), non-recurrent mortality (NRM), and overall survival (OS) in patients after allo-HSCT.This retrospective study included 222 patients with AML, myelodysplastic syndrome (MDS), acute lymphoblastic leukaemia (ALL), and chronic myelogenous leukaemia (CML) who underwent their first allo-HSCT at the Sun Yat-sen University’s First Affiliated Hospital between January 1, 2014, and March 31, 2021, with a follow-up cut-off date on June 30, 2021. Patients who had a history of autologous HSCT and those who received their stem cells from both cord blood and peripheral blood were not included in this study. Clinical and demographic information was gathered by reviewing medical records. Clinic visits, telephone conversations with families, and/or reviews of inpatient and outpatient clinical care records were used to gather overall survival data and follow-up information about complications. The First Affiliated Hospital of Sun Yat-sen University’s Ethics Committee gave the study approval, and it was carried out in compliance with the Helsinki Declaration. Two patients received their stem cells from both cord blood and peripheral blood, and one with a history of autologous HSCT were excluded. In total, 219 qualifying patients’ data were incorporated into the final analysis.The diagnosis of VZV infection was mainly based on specific clinical manifestations, which were defined by the detection of the VZV antigen or the emergence of typical cutaneous vesicular lesions . VZV reaThe primary endpoint of this study was CIR stratified by VZV reactivation. The secondary endpoints were the NRM and OS stratified by VZV reactivation. In this study, relapse after allo-HSCT referred to morphological relapse at any site as determined by the standard criteria. NRM was defined as mortality resulting from any reason other than relapse and was regarded as a competing risk for recurrence. OS was characterized as an interval from transplantation to the last follow-up date or death from any cause. Long-term survival was defined as OS ≥ 3 years. The starting point of follow-up was the date of allo-HSCT.Antiviral drugs should be administered prophylactically or preventively after allo-HSCT. To prevent infection of herpes simplex virus types 1 and 2, as well as varicella-zoster virus, valacyclovir was administered to these patients at least 6- 12 months after transplantation, and prolonged treatment course depended on the circumstances. If CMV DNAemia arises, replace valaciclovir with ganciclovir or foscarnet until DNA PCR for CMV is negative. For patients with VZV reactivation, valacyclovir (1000 mg 3 times daily) was given to these patients for 7 days , and theP ≤ 0.05 in univariate models were retained in the final model, and VZV reactivation was forced into the final model. The significance threshold for all bilateral tests was established at P < 0.05. The SPSS Statistics  and R 4.1.2 package cmprsk  software were used.Normally distributed measurement data were expressed as mean ± standard deviation, and the relevant data was analyzed using the independent samples t-test. The chi-square test was conducted to analyze categorical data that met the normal distribution. Non-normally distributed measurements and categorical data were analyzed using the Mann–Whitney U non-parametric test. The CIR and NRM stratified by VZV reactivation were calculated using Gray’s test, with NRM and relapse acting as competing risks. Fine and Gray’s proportional hazard model was employed to examine competing risks in a regression setting. The Kaplan–Meier technique was used to estimate OS probability, and the log-rank test was used to compare estimates. Hazard ratios (HRs) were estimated from the Cox regression models. Covariates included age of patients and donors, donor/recipient sex (mismatch vs. match), DRI (high risk or intermediate risk vs. low risk), HLA matching and donor relation, conditioning regimen (NMAC vs. MAC), the use of anti-thymocyte globulin (ATG) in the conditioning regimen (yes vs. no), DLI after transplantation (yes vs. no), VZV reactivation after transplant (yes vs. no), degrees of aGVHD (grade 2–4 vs grade 0–1), the occurrence of cGVHD (yes vs. no), and CMV reactivation after transplant (yes vs. no). All of the relevant risk factors were found using a forward-stepwise model selection method. Risk factors with The demographic and clinical data of all patients included in this study are summarized in Table P = 0.850). Additionally, 169 patients used ATG, with 41 (82.0%) patients with VZV reactivation and 128 (75.7%) patients without VZV reactivation (P = 0.463). Furthermore, 99 patients had cGVHD, with 28 instances (56.0%) occurring in patients with VZV reactivation and 71 instances (42.0%) occurring in patients without VZV reactivation (P = 0.113). No significant variations in the distribution of age, gender, disease type, DRI, pretreatment regimen, DLI usage, or incidence of CMV reactivation were found between the two groups. Between the two groups, the initial features were comparable  experienced VZV reactivation after allo-HSCT. As shown in Table P < 0.001) , 64 of the 219 patients (29.2%) relapsed following transplantation. As shown in Fig. , 64 of tP = 0.012). The CIR was 4.8% and 11.1% when VZV reactivation was present, and 31.3% and 51.8% when VZV reactivation was not present at 1 year and 3 years after the transplantation in ALL patients (P = 0.006). After adjusting for other covariates in multivariate competing-risks regression analyses by disease type     (Fig. In the entire cohort, the estimated 1- and 3-year estimated rates of OS were 75.9% and 65.4%, respectively. When stratified by VZV reactivation, the estimated rate of OS was 69.1% and 56.7% in patients without VZV reactivation, compared with 98.0% and 92.9% for those with VZV reactivation at 1 year and 3 years after the transplantation in the combined group (This investigation intended to explore the relationship between VZV reactivation and patients’ long-term prognoses following allo-HSCT, including CIR, NRM, and OS. In the combined cohort, VZV reactivation was significantly linked with decreased relapse, decreased NRM, and increased OS. The multivariate regression analysis confirmed that VZV reactivation was an independent protective predictor of CIR, NRM, and OS. For disease-type-based subgroup analysis, whether in AML or ALL patients, the multivariate regression analysis confirmed that VZV reactivation was still an independent protective predictor of CIR and OS. Our findings provide a way to lessen recurrence following allo-HSCT.The prognosis could be influenced by the patient's pre-transplant status, the transplant procedure, post-transplant complications, and donor status. In these two groups, the variations in patient demographic features, disease type, DRI, conditioning regimen, the incidence of aGVHD, the incidence of cGVHD, the incidence of CMV DNAemia and donor demographic characteristics were not statistically significant. Also, after multivariate analysis, VZV reactivation proved to remain significant and was the strongest risk factor. This boosted the reliability of the findings of this study.Whether VZV reactivation substantially contributes to the graft-versus-leukemic effect is unknown. However, it is worth investigating whether cellular immunity induced by VZV reactivation has an anti-leukemic effect.negγδ T-cells expand strongly and permanently, and these cells recognize leukemic blasts via their TCR, and CD8αα probably serves as a co-receptor in antigen recognition [Firstly, the reactivation of CMV, another herpes virus, may induce cellular anti-leukemic immunity . Much haognition , 15, 26.ognition , 28. Thiognition . Moreoveognition –9.Secondly, CMV infection may increase NRM which counteracts the benefit of the graft-versus-leukemic effect, and the graft-versus-leukemic effect may be influenced by several factors. A Japanese study involving 3,539 patients who underwent allo-HSCT demonstrated that CMV reactivation was significantly linked with reduced disease recurrence only in AML patients, but not in ALL, CML, or MDS patients. Additionally, CMV reactivation was strongly associated with increased NRM and overall mortality , 30. ButAdditionally, even in AML patients, Bao et al. discovered that in the ATG-using cohort, T-cell depletion diminished the anti-leukaemia effects induced by CMV reactivation . But in Therefore, it is plausible and worthy to hypothesize that VZV reactivation may directly contribute to anti-leukemic effects. To confirm this, prospective studies are urgently needed to assess the enduring immunological response of patients with VZV reactivation following allo-HSCT. It is reasonable to postulate that VZV reactivation directly contributes to the substantial long-term antileukemia effect reported in this study.In conclusion, after adjusting for confounding variables, this study revealed that VZV reactivation was linked to a significant decrease in CIR and NRM in patients following allo-HSCT and was an independent predictor of an excellent OS rate. This phenomenon of reduced leukaemia recurrence should be validated by several prospective studies, and the mechanisms underlying this anti-leukemic impact must also be confirmed by multiple prospective and experimental research.


Pharmacy benefit managers (PBMs) play a major role in administering prescription drug benefit programs for health plans and employers. PBMs have often encouraged the use of self-owned mail-order pharmacy services with the promise to plan sponsors of lower prescription drug costs compared with those of the community pharmacy network. Some plan sponsors have requested a higher degree of disclosure of contract relationships and transparency in pricing. Unfortunately, little research exists based on empirical data to determine the net plan cost and member cost for mail-order drugs, as opposed to having these drugs dispensed by community pharmacies.To determine the difference between mail-order and community pharmacy in the (1) payment (cost) per day of drug therapy for the plan sponsor and for the member for the highest expenditure therapeutic classes, (2) generic dispensing ratios for all drugs and for a comparative market basket of drugs, and (3) cost per unit for the top 20 generic drugs dispensed through the mail-order channel.Pharmacy claim records were obtained from 2 publicly financed pharmacy benefit plans in Texas for fiscal year 2004 . There were approximately 460,000 members in Plan A and 177,000 members in Plan B. Pharmacy cost per day  was calculated for each drug in the 30 highest expenditure therapeutic categories and aggregated for mail-order and community pharmacy channels for each plan. Differences in the mail-order and community pharmacy cost per day were calculated for each drug (adjusted for dosage) in the therapeutic category and weighted by the products' share of mail-order therapy days within the therapeutic category. A weighted cost per day for each therapeutic category was calculated with a comparison of what the cost would have been for plan cost and member cost if all mail claims had been paid based on the community pharmacy cost per day. Comparison of the cost per day helped control for differences in quantity dispensed per day per product and for product mix within each therapeutic category. Descriptive analyses were conducted to compare generic dispensing ratios between (1) all mail-order and community pharmacy claims, and (2) a market basket of therapeutic categories most commonly found within the mail-order channel. Finally, the difference in price per unit was calculated between mail-order and community pharmacy channels for the top 20 generic drug products.Mail-order drugs accounted for 34.4% of overall pharmacy benefit spending, including plan cost and member cost, in Plan A and 43.4% for the market basket of drugs compared with 56.0% of overall spending and 63.1% for the market basket in Plan B. When comparing the cost per day for the top therapeutic categories, the authors found the plan sponsor cost was higher for mail-order than for the community pharmacy channel for approximately half of the top therapeutic categories. This result contributed to a 0.5% higher plan cost per day for mail-order ($1.24) than for community pharmacy ($1.23) for Plan A but a 0.4% lower plan cost per day for Plan B ($1.43 for mail-order vs. $1.44 for community pharmacy). The member cost was lower for mail-order than for community pharmacy for almost every therapeutic category, and overall was 29% lower in Plan A ($0.73 per day for mail-order vs. $1.03 for community pharmacy) and 37% lower in Plan B ($0.52 for mail-order vs. $0.82 for community pharmacy). For all claims, the generic dispensing ratios were lower in the mail-order channel than in the community pharmacy channel (37.7% vs. 49.0% for Plan A and 34.7% vs. 45.0% for Plan B). The cost per unit  for the top 20 generic drug products dispensed by mail order was 16.5% lower than community pharmacy for the plan sponsor in Plan A but 18.0%.


While social media is increasingly used by dermatologists to educate the public , any socTikTok and Instagram were selected for their size and dearth of published literature, compared to the known presence of misinformation on platforms like Twitter and Facebook . On TikTNondermatologists made up the majority  of telangiectasia-related content on TikTok . Of 123 Many patients use social media platforms for dermatologic information . Our finThis study provides a sample of content creators in telangiectatic-related content. This study reinforces the importance of social media presence of board-certified dermatologists to comment on and combat inaccuracies by creating educational content and reacting to erroneous information. Further research is necessary to evaluate the scope of misinformation and its deleterious effects.


The polyethylene glycol (PEG) additive adsorbed to the friction material surface can keep the shear plane away from the solid surface, reducing the absolute zeta potential and EOF. The electroosmotic performance of cutting fluid can be improved by compounding additives with different electroosmotic performance functions. Furthermore, electroosmotic regulators can adjust the zeta potential by the electrostatic adsorption mechanism, affecting the penetration performance of cutting fluid in the capillary zone at the friction interface. The improvement in the tribological performance of cutting fluid developed with consideration given to the electroosmosis effect is attributed to the enhancement of the penetration ability of the cutting fluid and the formation of more abundant amounts of lubricating film at the interface.The electroosmosis effect is a complement to the theory of the traditional capillary penetration of cutting fluid. In this study, based on the electric double layer (EDL) characteristics at friction material/solution interfaces, the influences of additives and their concentrations on capillary electroosmosis were investigated, and a water-based cutting-fluid formulation with consideration to the electroosmosis effect was developed. The lubrication performance levels of cutting fluids were investigated by a four-ball tribometer. The results show that the EDL is compressed with increasing ionic concentration, which suppresses the electroosmotic flow (EOF). The specific adsorption of OH Cutting fluids play an important role in machining by providing lubrication, cooling, cleaning, and rust prevention to improve the surface quality of workpiece and extend tool life . Researc2 nanofluid lubricant at the steel–steel and steel–ceramic friction interfaces and improve the tribological performance of the lubricant. Generally, the zeta-potential characteristics (amplitude and polarity) depend on the physical and chemical properties between solid surface and liquid. Falahati et al. -1-propanesulfonate  and cetyltrimethylammonium bromide  on the tribological performance levels of cutting fluid at the steel–steel interface were investigated by four-ball tribometer. The penetration ability of cutting fluid was evaluated by elemental analysis of worn surfaces.4K2O7·4H2O) or fatty alcohol polyoxyethylene ether phosphate nPO(OH)2 and [RO(CH2CH2O)n]2PO(OH)) EP additive solutions , and triethanolamine (TEA) pH buffer solutions . The determination of additive concentration levels was based on the common proportions of additives in water-based cutting fluids [Lubricity additive, EP additive, and pH buffer were selected to form the basic formula of a water-based cutting fluid. Additionally, CHAPS and CTAB were selected as electroosmotic regulators to further optimize the electroosmotic performance of the basic formula. Information on the chemical reagents used is listed in g fluids . AccordiP on both sides of the microchannel was adjusted by a pressure regulator, which increased linearly from 0.02 MPa to 0.1 MPa. The potential difference between the two sides of the microchannel  was measured by an Agilent 34420A voltmeter . According to the streaming potential coefficient (SE/∆P), the zeta potential at the friction material/solution interface was calculated as follows:ζ is the zeta potential, η is the solution viscosity, ε = ε0rε is the solution permittivity, and λ is the solution bulk conductivity.The tangential streaming potential measurement technique is widely used to determine the zeta potential at the solid/liquid interface because the calculated zeta potential more accurately reflects the EDL characteristics at the interface ,48. FiguL, W, and H are the length, width, and height of the microchannel, respectively, corresponding to a size of 160 × 10 × 0.3 mm; and tG is the total conductance of the AISI 52100 steel microchannel filled with solution; and tG = 1/tR. tR is the total resistance of the AISI 52100 steel microchannel filled with solution, as measured by the Keithley 6517B electrometer.Due to the excellent electrical conductivity of AISI 52100 steel, numerous ions flow back through the material bulk when measuring the streaming potential at the AISI 52100 steel/solution interface . TherefoEDL conductivity was used in this study to calculate the zeta potential at the alumina ceramic/solution interface [EDLλ is the EDL conductivity of the alumina ceramic microchannel filled with solution, and the galvanostatic four-electrodes system combined with electrochemical impedance spectroscopy was used for measurement and calculation.In addition, the absolute value of the zeta potential at the alumina ceramic/solution interface would be underestimated by Equation (1) in the case of a solution with low ion concentration. Therefore, the nterface :(3)ζ=ESΔThe MMW-1 multi-specimen test system  was used to conduct the four-ball friction test under full immersion lubrication. The specific test conditions are listed in The formation of EOF is caused by the action of the axial electric field on counterions in the EDL. Hence the amplitude and polarity of the zeta potential can considerably impact the magnitude and direction of EOF. To further investigate the influences of additives and their concentrations on capillary electroosmosis, PEG400, MOA-3P, and TEA were selected to test in order to calculate their streaming potential and zeta potential. The streaming potentials of the three additive solutions on the surface of AISI 52100 steel and alumina ceramic are all negative, as shown in tR of AISI 52100 steel microchannels filled with these solutions and the EDL conductivities EDLλ of alumina ceramic microchannels filled with these solutions are listed in The total resistance The relationship between the zeta potential and the concentration of the PEG400 solution is depicted in Based on the investigation of the EOF velocities and zeta potentials of PEG400, MOA-3P, and TEA solutions, it is clear that the electroosmotic performance levels of the three additive solutions are quite different. The EOF velocity of TEA is higher than that of deionized water, while the EOF velocity of PEG400 is lower than that of deionized water. Additionally, the EOF velocity of MOA-3P is higher than that of deionized water at low ion concentrations and lower at high ion concentrations. PEG400 and TEA concentrations in the water-based cutting fluid were set at 3 wt% and 3 wt%, respectively, in consideration of the electroosmotic performance of the additives and their content level in the general formula ,61. ThentR and the EDL conductivities EDLλ MOA-3P and TEA are used as EP additive and pH buffer, respectively, for water-based cutting fluid. Their excessive ion concentration will lead to a compression of EDL at the friction material/solution interface, causing the absolute value of the zeta potential and EOF to decrease.(2)PEG400 is used as a lubricity additive for water-based cutting fluid. Its chain segments are adsorbed to the surface of friction material to produce steric hindrance, which makes the shear plane move away from the solid surface, and the absolute value of zeta potential and EOF decrease.(3)− ions on the AISI 52100 steel surface and the dissociation of Al-OH groups on the alumina ceramic surface can be promoted to some extent by an increase in pH value in a water-based cutting-fluid system, which increases the absolute value of zeta potential and EOF.The specific adsorption of OH(4)The electroosmotic performance of water-based cutting fluid can be further adjusted by adding electroosmotic regulators. The electroosmotic promoter CHAPS increases the absolute value of zeta potential by electrostatic adsorption, which promotes the penetration ability of cutting fluid in the capillary zone of the friction interface, improving the tribological performance of cutting fluid at the steel–steel interface. The electroosmotic suppressant CTAB reduces the absolute value of the zeta potential and suppresses EOF, exhibiting poor tribological performance.In this paper, the EOF velocities and zeta-potential characteristics of water-based cutting-fluid additives and their compound cutting fluids were investigated. And the mechanisms of additives and compound cutting fluids for electroosmotic performance were revealed, which provides a reference for the formulation design of cutting fluids based on the electroosmosis effect. In addition, the effects of electroosmotic regulators on the tribological performance of cutting fluid were verified. The main conclusions are as follows: