Ex vivo culture to evaluate dynamic changes in the tumor microenvironment following durvalumab treatment

Ex vivo culture to evaluate dynamic changes in the tumor microenvironment following durvalumab treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ex vivo culture to evaluate dynamic changes in the tumor microenvironment following durvalumab treatment Typhaine Gris, Heidi Shore, Claire Ménard, Louis Lacombe, Vincent Fradet, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7180379/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While the tumor microenvironment (TME) is critical to the mechanism of action of anti-PD-1/PD-L1 checkpoint inhibitions, limitations exist to understand this using in vitro and in vivo models. With bladder (BCa) and prostate (PCa) cancer representing cancer with respectively strong and poor responses to PD-1/PD-L1 inhibition, we used ex vivo culture of fresh patient cancer tissue to better understand changes in the TME following durvalumab therapy, specifically the macrophage phenotype. Fresh prostate or bladder biopsies from 70 patients were cultured with durvalumab or isotype control. Flow cytometry assessed a panel of myeloid cell markers. In BCa samples, there was a significant difference, after durvalumab treatment, in PD-L1 expression on non-immune cells and macrophages and increased reactive oxygen species (ROS) production by myeloid cells in BCa. In PCa samples, there was a significant decrease in PD-L1 expression on non-immune and immune cells and increased ROS production by macrophages. Concomitantly, we observed a significant decrease in phagocytosis activity in immune cells and macrophages in prostate biopsies. Our detailed cytometric analysis of ex vivo cultured BCa and PCa biopsies demonstrated changes in macrophage phenotype after durvalumab treatment. This study shows ex vivo culture can ascertain trends in tumor immune cell responses to immunotherapy. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology Health sciences/Urology bladder cancer prostate cancer checkpoint inhibition myeloid cells tumor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Checkpoint inhibition immunotherapy represents a significant paradigm shift in the treatment of many cancers, including bladder cancer (BCa). However, for others, it remains largely ineffective, such as in prostate cancer (PCa). These differences may be partly explained by genomic factors, with BCa recognized to have a very high tumor mutational burden (TMB) in contradistinction to PCa 1 . Up to 55% of the variation in efficacy of PD-1 inhibition has been estimated to be attributable to TMB 2 . Thus, other factors within the tumor microenvironment (TME) are significant contributors to influence response rates to immune checkpoint blockade. Understanding these differences and the heterogeneity between cancers represents an opportunity to identify immune resistance mechanisms which may be targeted to improve the proportion of responders. The TME is made of heterogenous cancer cells, stromal cells and immune cells, as part of a complex ecosystem 3 . The continuum of cancer immune surveillance, as well as its natural history and it’s receptivity to treatment, are defined by the spatial distribution and functional orientation of immune cells infiltrating the tumor 4 . Immune checkpoints such as PD-1/PD-L1 negatively regulate the immune system, are present on various cells including macrophages 5 , 6 . The PD-1/PD-L1 system may attenuate local T cell activation, proliferation, survival and cytotoxic secretion within cancer and decrease tissue damage 7 , 8 . Macrophages are often conceptually dichotomized as M1 or proinflammatory macrophages and M2 anti-inflammatory macrophages 9 . Prior studies suggest a predominance of immunosuppressive myeloid cells is associated with poorer responses to anti-PD-1 therapy 10 – 14 . Durvalumab is a fully human monoclonal antibody targeting PD-L1 from binding to its receptors PD-1 and CD80. IMFINZI (Durvalumab for injection) is currently approved for clinical use in urothelial carcinoma 15 and muscle invasive bladder cancer 16 in Canada. A trial in muscle-invasive bladder cancer has shown event-free and overall survival benefits when combining durvalumab with chemotherapy 17 . Understanding the changes which occur in the human TME following treatment with durvalumab may provide insights to improve treatment strategies. We performed a prospective study using ex vivo culture of biopsies from prostates and bladders that were surgically removed to investigate how durvalumab influences the phenotype of macrophages within the respective cancerous organs. The use of ex vivo culture represents an attractive model in that it permits direct evaluation of cells within the TME 18 – 24 . Given the explanted nature precludes the infiltration of immune cells and our prior experience, we focus on changes in myeloid cells after 72h of treatment 25 . The objective was to evaluate phenotypic changes in myeloid cells within the microenvironment of bladder and prostate tumors after durvalumab treatment. We additionally investigate for relationships between immune myeloid cell phenotypes and patient and tumor characteristics. Methods Patient recruitment. Institutional research ethics committee (REC) approval was obtained from the CHU de Québec-Université Laval (#2019–4181 and #2020–4547). Patients undergoing radical cystectomy or radical prostatectomy for urothelial carcinoma or prostatic adenocarcinoma, respectively, were targeted for this study. Informed consent was obtained from all patients who contributed fresh samples to our URO-1 institutional biobank (#2012 − 1002), and all methods complied with applicable ethical guidelines and regulations. Seventy patients were recruited for this study (35 bladders and 35 prostates). For BCa patients, the inclusion criteria were age ≥18 years, the ability to give informed consent, histologically confirmed urothelial carcinoma with a scheduled radical cystectomy, visibility of the tumor on pre-operative imaging or history of multiple tumors. Patients with or without pre-operative chemotherapy were included. The exclusion criteria for BCa patients were aborted surgery, inadequate tissue obtainable from the pathologist, known autoimmune disorders, prior anti-PD-1/PD-L1 or similar immune checkpoint inhibition within 2 years, receipt of high-dose steroids within 2 weeks of surgery and non-urothelial carcinoma histology. For PCa patients, the inclusion criteria were age ≥18 years, ability to give informed consent, histologically confirmed prostate adenocarcinoma and a scheduled radical prostatectomy. The exclusion criteria for PCa patients were aborted surgery, inadequate tissue obtainable from the pathologist, known autoimmune disorders, patients who have received anti-PD-1/PD-L1 or similar immune checkpoint inhibition within 2 years, receipt of high-dose steroids within 2 weeks of surgery, Gleason grade group 1 prostatic adenocarcinoma or non-adenocarcinoma histology. Bladder and prostate biopsy procurement. Biopsies for BCa were performed with 18G biopsies in the tumor area and a 5mm dermatological punch in the normal area. Biopsies for PCa were performed with 18G biopsies in the tumor area and in the normal area (1/3, 2/3 ratio) (Fig. 1 A). Ex vivo culture of biopsies. Technique was based on our previous work with PCa biopsies 26 , 27 , with optimisation studies to select the optimal methods for bladder tissues. As previously described, bladder and prostate biopsies were first washed with HBSS Ca 2+ Mg 2+ (#311-513-CL, Wisent Bioproducts, St-Bruno, QC, Canada). Washed biopsies were then cut into two pieces; punches were cut in four pieces directly on plate with HBSS Ca 2+ Mg 2+ to keep tissues moist, with both pieces separated in a matched manner between isotype control (AstraZeneca, Cambridge, UK, 20µg/ml) and durvalumab (AstraZeneca, 20µg/ml) treatment for 72h. That is, a sample of normal adjacent tissue and of tumor sample were placed in each condition to balance comparisons. The biopsies were cultured in DMEM-F12 (#319-090-CL, Wisent Bioproducts) with 10% autologous serum obtained immediately prior to surgery, Glutamax (#35050061, ThermoFisher Scientific, Ottawa, ON, Canada, 1000mg/ml), HEPES (#15630080, Sigma-Aldrich, Saint-Louis, MO, 25mM) and Primocin (#ANT-PM-05, InvivoGen, San Diego, CA, 10ml/L) at 37°C with 5% CO 2 . Tissue dissociation for cytometry analysis. For BCa, biopsies were minced manually and using a GentleMACs dissociator (Miltenyi Biotec, San Diego, CA). Dissociated tissues were incubated with Collagenase III (#LS004182, Wisent Bioproducts, 10µl/10ml) for 30 min of agitation at 37°C followed by DNAse 1 (#260913, Sigma-Aldrich, 10µl/10ml) for 5 min of agitation at 37°C. The cellular suspension was filtered with a 70µm cell strainer. For PCa biopsies, the protocol was the same as the protocol for BCa except that Collagenase II (#17101015, Thermo Fisher, 1µl/ml) and DNAse 1 (Sigma-Aldrich, 1µl/ml) were added prior to overnight incubation. Accutase (#25-058-CL, Corning, NY) was also subsequently added to prostate biopsies, with a 70µm cell strainer used to remove clumps. Cell staining and flow cytometry analysis. After dissociation, cells were incubated 10 min at room temperature with Fc block (#564219, BD Biosciences, Mississauga, Ontario, Canada) and then stained with an extracellular antibody panel for 30 min at 37°C, for 20 minutes for permeabilization and for 30 min at 4°C in the dark for intracellular antibodies. Antibodies used are detailed in Supplementary Table 1. Cell viability was assessed using the Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA). Stained cells were acquired on a LSR Fortessa cytometer (BD Immunocytometry Systems, San Jose, CA) and data collection was performed using BD FACS Diva software (BD Bioscience). Data analyses were performed using FlowJo software (v.10.7.1, FlowJo, LLC). Clinical correlation analyses. Chart review identified pre-operative characteristics of each patient, including laboratory, pathological, and clinical data, similar to previous work 25 . The pre-prostatectomy CT scan distance between the prostate and pubic symphysis was used to quantify the periprostatic fat. We choose to use the minimal measured distance and dichotomized this by the median 28 into high and low periprostatic fat categories. Statistical analyses. Descriptive statistics evaluated immune cell composition of biopsies according to patient and tumor characteristics. A paired T-test evaluated differences in the relative expression of cells markers between isotype control and durvalumab treatment conditions in bladder and prostate biopsies. As an exploratory study, no correction was performed for multiple testing. Analyses were performed using GraphPad Prism 9.5.1. For clinical characteristics of the BCa and PCa cohort, means are presented ± standard deviation unless otherwise indicated. Results Clinical characteristics. Clinical characteristics of the BCa cohort and the PCa cohort are summarized in Table 1. For BCa the mean age was 67.7 ± 10 years, 15 patients received neoadjuvant chemotherapy, the mean patient weight was 74.5 ± 14.2 kg and the mean body mass index (BMI) was 26.0 ± 4.3. For PCa, the mean age was 66.7 ± 6 years, the mean weight was 79.2 ± 12.8 kg and the mean BMI was 26.4 ± 3.8. Ex vivo culture to evaluate changes in immune cells within the BCa microenvironment. In bladder samples we observed no significant change in the proportion of live cells following 72h of ex vivo treatment between durvalumab and isotype control (Figure 1B). Durvalumab decreased the proportion of PD-L1 expressing non-immune cells (CD45 - PD-L1 + cells, Figure 2A) and macrophages (CD45 + CD11b + HLADR + PD-L1 + cells, Figure 2B) in bladder samples. Further, there was a significant increase of reactive oxygen species (ROS) production by myeloid cells (CD45 + CD11b + ROS + cells, Figure 2C) after durvalumab treatment. No other significant differences were observed among both macrophage and general immune cell markers. This non-significant change in markers expression is represented in tSNE analyses on macrophages of all BCa patients treated with isotype or durvalumab (Supplementary Figure 1A-B). Ex vivo culture to evaluate changes in immune cells within the PCa microenvironment. Similarly, among the prostate biopsies, we observed good cell viability after 72h of treatment (Figure 1C). Further, in the prostate biopsies, durvalumab once more decreased the expression of the immune checkpoint PD-L1 in non-immune cells (CD45 - PD-L1 + cells, Figure 3A) and in immune cells (CD45 + PD-L1 + cells, Figure 3B). Among the macrophage population (CD45 + CD11b + HLADR + cells), we observed that expression of ROS increased following durvalumab treatment compared to isotype control (Figure 4A). We also observed a concomitant decrease in the proportion of phagocytic immune cells (CD45 + Phago + cells, Figure 4B) and phagocytic macrophages (CD45 + CD11b + HLADR + Phago + cells, Figure 4C). Furthermore, the mean fluorescence intensity (MFI) of CD45 + Phago + cells and CD45 + CD11b + HLADR + Phago + cells were also significantly lower (data not shown). No other significant differences were observed among both macrophage and general immune cell markers. Likewise, tSNE analyses demonstrate non-significant changes in markers expression in macrophages of all PCa samples (Supplementary figure 1C-D). Relation between the macrophage-associated markers and clinical characteristics. Using our above results in the isotype-control group, we next assessed for correlation between macrophage-lineage immunosuppressive markers (CD45 + CD11b + HLA-DR + cells) and specific clinical characteristic of patients (Table 1). For BCa patients (Table 2), there were no significant correlations between the individual expression of selected cell cytometry profiles and selected clinical characteristics, except for a negative correlation between CD206 + macrophage expression and the presence of lymphovascular invasion (LVI, r=-0.3428; *p=0.0438), and a negative correlation between the PD-L2 macrophage expression and patient weight (r=-0.3465, *p=0.0415) or BMI (r=-0.5028, *p=0.0021). For the same analysis in PCa patients (Table 3), we observed a positive correlation between the CD206 + macrophage expression and the presence of intraductal carcinoma of the prostate (IDC, r=0.3646, *p=0.0313), between the PD-L1 macrophage expression in macrophages and the prostate tumor volume (PTV, r=0.335, *p=0.0492), and between macrophage ROS production and periprostatic fat (PF, r=0.461, *p=0.0354). In fact, ROS production by macrophages (CD45 + CD11b + HLADR + ROS + cells) is significantly increased by durvalumab treatment in patients with low periprostatic fat whereas it is unchanged after durvalumab treatment in patients with high periprostatic fat (Supplementary figure 2). We also found a negative correlation between the abundance of macrophages and the presence of perineural invasion (PNI, r=-0.3677, *p=0.0456), between the CD163 + macrophage expression and seminal vesicle invasion (SVI, r=-0.4337, *p=0.0092), and between the PD-L1 macrophage expression and PNI (r=-0.5001, *p=0.0049) Discussion Our results using detailed flow cytometry demonstrate both the limits and utility of ex vivo culture to evaluate dynamic changes in myeloid cell markers. Our results demonstrate evidence of an expected decrease in PD-L1 expression following anti-PD-L1 treatment with durvalumab in both PCa and BCa patients. However, of the different immune phenotype markers evaluated, most changes were observed were only for functional markers: ROS production and phagocytosis activity. Moreover, we overall observed more significant changes in the prostate biopsies than the bladder samples. Notably, we showed that our culture methods did not affect cell viability after 72h, highlighting the utility for both other treatments and other tissue types. The observed decrease in PD-L1 expression on both immune and non-immune cells in prostate biopsies and in non-immune cells and macrophages in bladder samples suggests that durvalumab interacted with individual cells as expected during ex vivo treatment. With our cells washed significantly prior to flow cytometry, it is unlikely the decreases are related to durvalumab interfering with binding of antibody panel anti-PD-L1. We can infer that this decrease may have functional consequences. Prior research has suggested macrophage expression of PD-L1 contributes to active removal of T cells from the TME, suggesting a role for myeloid cells and PD-L1 inhibition in the trafficking of T cells in the TME 29 . We also observed a decrease of the PD-L1 expression in non-immune cells in both BCa and PCa samples following durvalumab treatment. It is a reasonable inference that most of these cells are cancer cells. Cancer cell expression of PD-L1 can directly suppressing the cytotoxic activity of PD1 + T cells 30 , though we did not evaluate T cells in our study. Our novel finding of an increase in ROS production and a decrease of phagocytosis activity in macrophages in prostate biopsies suggests PD-L1 inhibition may induce a more favorable M1 macrophage phenotype within the prostate. These results also suggest a switch in macrophage phenotype (anti-inflammatory toward pro-inflammatory) in response to durvalumab. In our explorations of correlations between clinical characteristics and macrophages phenotypes we did not validate our prior findings with prostate biopsies 25 . However, the positive correlation between the presence of IDC and the presence of M2 macrophages (CD45 + CD11b + HLA-DR + CD206 + cells) did resemble our prior association of IDC with another (B7-H3 + ) immunosuppressive macrophage phenotype 31 . Interestingly, macrophage ROS production was positively correlated with the amount of periprostatic fat, suggesting a mechanism related to its link with more aggressive PCa 32 , 33 . In contrast to recent work in mouse PCa models suggesting a link between tumor-associated macrophage PD-1/PD-L1 expression and obesity-associated signaling 34 , we observed no correlation in human prostates between BMI and the presence of PD-L1 + macrophages. The strengths of this study include the use of fresh patient samples to analyze the TME and the use of optimized flow cytometry to provide detailed information on cell and functional markers following durvalumab treatment. Nonetheless, as with all studies assessing patient samples, heterogeneity between the patients concerning their individual and tumor characteristics is confounding. Overall, our results suggest heterogeneity was greater among BCa patients. Similarly, while every effort was made to pair samples between treatments, tissue heterogeneity adds further limitations. Nonetheless, our methods yielded high cell viability and quality flow cytometry results. However, we only evaluated changes after 72h of treatment. The time frame for changes to the different markers evaluated is not known and this may explain why the more functional markers of ROS production and phagocytosis had the most detectable differences. Finally, our study was focused on macrophages, without data on other pertinent cells such as T cells. In summary, our detailed cytometric assessment of samples from BCa and PCa patients reveal the limits and utility of ex vivo treatment assessments of the TME. While observing expected changes in PD-L1 for both samples, relatively few overall changes were observed. Nonetheless, we notably observed an increase in myeloid ROS production in BCa and a similar increase in macrophage ROS production in PCa. Further, in PCa, a concomitant decrease in macrophage and immune cells phagocytosis was present. Together, our direct analyses of both tumor and adjacent normal tissue highlights the opportunities and challenges to capture overall trends in individual immune cells responses to treatment despite tissue and patient heterogeneity. Declarations Acknowledgments The authors thank Zohra Berrehail, Chloé Miranda, Maryke Venne and Geneviève Trépanier for their contributions to this project. Author responsibilities: Conception and design: TG, PT Acquisition of data: TG, HS, CM Analysis and interpretation of data: TG, HS, PT Drafting of the article: TG, HS Article revision for critically for important intellectual content: LL, VF, YF, FP, ML Statistical analysis: TG, HS, PT Obtaining funding: PT Administrative, technical or material support: TG, CM Supervision: TG, PT Data availability: The data that support the findings of this study are available from the corresponding author upon reasonable request. Potential conflicts of interest: PT reports research funding from AstraZeneca as well as personal fees as a consultant for Bayer, Knight, TerSera, and Abbvie. Other authors have no potential conflicts of interest to report. Funding: This study was supported by an externally sponsored research grant from AstraZeneca (ESR-19-20213, PT). PT also is supported by a clinician-scientist award from Fonds de Recherche du Québec – Santé (#354004). Author Contribution Conception and design: TG, PTAcquisition of data: TG, HS, CMAnalysis and interpretation of data: TG, HS, PTDrafting of the article: TG, HSArticle revision for critically for important intellectual content: LL, VF, YF, FP, MLStatistical analysis: TG, HS, PTObtaining funding: PTAdministrative, technical or material support: TG, CM Supervision: TG, PT References Martincorena, I. & Campbell, P. J. Somatic mutation in cancer and normal cells. Science (New York, N.Y.) 349 , 1483-1489, doi:10.1126/science.aab4082 (2015). Yarchoan, M., Hopkins, A. & Jaffee, E. M. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. The New England Journal of Medicine 377 , 2500-2501, doi:10.1056/NEJMc1713444 (2017). Lee, P. P. & Marincola, F. M. Cancer treatment and research; v. 180; volume 180 (Springer, Cham, 2020). Galon, J., Angell, H. K., Bedognetti, D. & Marincola, F. M. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. 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Bladder cancer patients (n=35) Prostate cancer patients (n=35) Age (years) 67.74 ± 10 66.69 ± 6.2 Surgical Pathology Gleason Group Grade 2 - 15 3 - 8 4 - 4 5 - 8 Group Grade N/A 9 - High 26 - T Stage N/A 1 - pTis 5 - pTa 5 - pT0 6 - pT1 4 - pT2 - 12 pT2b 4 - pT3a 4 16 pT3b 4 7 pT4 2 - Extraprostatic extension (EPE) - 23 Intraductal carcinoma (IDC) - 14 N Stage Negative 25 28 Positive 8 7 N/A 2 - Surgical Margin status Negative 28 18 Positive 5 17 N/A 2 - Mean % Prostate Tumor Volume (min-max) 17.46 (1-80) Table 2: Correlation between characteristics of BCa disease and macrophages associated markers in the macrophage population. Spearman’s rank correlation table show the correlation between specific characteristics of the BCa and the proportion of macrophages (CD45 + CD11b + HLADR + cells) expressing M1 markers (CCR7, ROS expression), M2-associated markers (CD163, CD206) or immune checkpoints (B7H3, PD-L1 or PD-L2). Correlation coefficients (r s ) <-0.3 with significant p-values are indicated in bold. NLR pre-operative neutrophil-to-lymphocyte ratio, LVI Lymphovascular invasion, BCG Bacillus Calmette-Guerin treatment, NAC Neoadjuvant chemotherapy, PM Positive margin, W Weight and BMI Body mass index. CD45 + CD11b + HLADR + CD45 + CD11b + HLADR + B7H3 + CD45 + CD11b + HLADR + CCR7 + CD45 + CD11b + HLADR + CD163 + CD45 + CD11b + HLADR + CD206 + CD45 + CD11b + HLADR + PD-L1 + CD45 + CD11b + HLADR + PD-L2 + CD45 + CD11b + HLADR + ROS + NLR -0,09766 0,009456 -0,1713 0,06755 -0,1458 0,02809 -0,204 0,250 LVI 0,008304 -0,07634 -0,07839 -0,002393 -0,3428 0,03364 0,2177 -0,04159 BCG -0,09964 0,04501 -0,06345 -0,05452 -0,06961 -0,0684 -0,1286 -0,1303 NAC 0,1098 -0,2491 -0,1732 -0,1256 0,08728 -0,2855 -0,1575 0,2872 PM 0,1095 -0,05635 -0,01759 -0,01306 0,08557 0,08888 0,1613 0,08809 W 0,01397 -0,02255 -0,06345 0,06262 0,3134 -0,09716 -0,3465 0,1841 BMI -0,06914 0,06666 0,1462 0,1074 0,3041 -0,06878 -0,5028 0,316 Table 3: Correlation between characteristics of PCa disease and macrophages associated markers in the macrophage populations. Spearman’s rank correlation table show the correlation between specific characteristics of the PCa disease and the proportion of macrophages (CD45 + CD11b + HLADR + cells) expressing M1 markers (CCR7, ROS expression), M2-associated markers (CD163, CD206) or immune checkpoints (B7H3, PD-L1 or PD-L2). Correlation coefficients (r s ) >0.3 or <-0.3 with significant p-values are highlighted in bold. NLR Neutrophil-to-lymphocyte ratio, PSA Prostate specific antigen, EPE Extraprostatic extension, SVI Seminal vesicle invasion, IDC Intraductal carcinoma of the prostate, PNI Perineural invasion, LVI Lymphovascular invasion, PTV Prostate tumor volume, PM Positive margin, W Weight, BMI Body mass index and PF Periprostatic fat. CD45 + CD11b + HLADR + CD45 + CD11b + HLADR + B7H3 + CD45 + CD11b + HLADR + CCR7 + CD45 + CD11b + HLADR + CD163 + CD45 + CD11b + HLADR + CD206 + CD45 + CD11b + HLADR + PD-L1 + CD45 + CD11b + HLADR + PD-L2 + CD45 + CD11b + HLADR + ROS + NLR 0,7339 0,01203 -0,004516 0,1504 0,2386 -0,2509 -0,07434 -0,05958 PSA -0,1618 0,003981 0,06091 0,1841 -0,05719 0,002978 -0,1145 -0,02607 EPE -0,2732 0,1034 0,1224 -0,2944 0,2193 0,0511 0,07172 0,01983 SVI -0,2271 0,2873 0,1715 -0,4337 0,1189 0,273 0,1088 0,08788 IDC -0,1038 0,1055 0,1272 -0,1284 0,3646 0,1373 0,1455 0,1915 PNI -0,3677 -0,2358 -0,07885 -0,2403 -0,04449 -0,5001 0,1231 0,2033 LVI 0,3151 0,1002 0,3756 -0,165 -0,3279 0,2397 -0,3027 0,2789 PTV -0,05024 0,06082 -0,2459 -0,1356 0,05464 0,335 0,4933 -0,1008 PM -0,2889 0,09557 -0,02476 -0,1933 0,03092 0,07537 0,04544 0,06409 W -0,233 0,0303 0,04427 -0,0467 -0,07282 -0,03971 -0,09311 0,04511 BMI -0,1659 0,1554 0,03461 -0,248 0,09693 0,0793 0,07944 0,02048 PF -0,2787 -0,04275 -0,01637 -0,02786 -0,01484 0,06437 0,002367 0,461 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7180379","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":494707491,"identity":"77b933aa-8746-4039-8868-a30baf548a9e","order_by":0,"name":"Typhaine Gris","email":"data:image/png;base64,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","orcid":"","institution":"Université Laval","correspondingAuthor":true,"prefix":"","firstName":"Typhaine","middleName":"","lastName":"Gris","suffix":""},{"id":494707493,"identity":"643cc449-122b-4f1f-8e24-4f8aa8bb67fd","order_by":1,"name":"Heidi Shore","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Heidi","middleName":"","lastName":"Shore","suffix":""},{"id":494707495,"identity":"e3d24ff9-ce12-49b0-9ce8-3b0457e9bc04","order_by":2,"name":"Claire Ménard","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Ménard","suffix":""},{"id":494707497,"identity":"ba56aeaa-3695-4e90-b613-be46f1fc8115","order_by":3,"name":"Louis Lacombe","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Louis","middleName":"","lastName":"Lacombe","suffix":""},{"id":494707500,"identity":"38925bf1-0d5d-4dd0-ad4a-8c3cfb207020","order_by":4,"name":"Vincent Fradet","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Fradet","suffix":""},{"id":494707502,"identity":"dc94744c-1885-441d-b685-bb0fc010a1fa","order_by":5,"name":"Frédéric Pouliot","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Pouliot","suffix":""},{"id":494707504,"identity":"2b79ae80-2c6f-49af-ad07-1e64d14f844d","order_by":6,"name":"Michele Lodde","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Lodde","suffix":""},{"id":494707505,"identity":"c24a6215-dfc0-436f-b659-86420f23c5e5","order_by":7,"name":"Yves Fradet","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Yves","middleName":"","lastName":"Fradet","suffix":""},{"id":494707506,"identity":"cb5d69de-cc43-4ba6-a0e1-fe981429bbe4","order_by":8,"name":"Paul Toren","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Toren","suffix":""}],"badges":[],"createdAt":"2025-07-21 19:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7180379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7180379/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88648053,"identity":"f2848a65-5bed-408f-9b3c-29774b56565a","added_by":"auto","created_at":"2025-08-08 16:53:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":350130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of bladder and prostate cancer biopsies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eSchematic representation of the steps to analyze the TME of BCa and PCa. Schema done with the help of BioRender.com. The proportion of live cells after dissociation following 72h of \u003cem\u003eex vivo\u003c/em\u003e culture was similar between durvalumab treatment and isotype control for \u003cstrong\u003eB. \u003c/strong\u003eBCa samples and \u003cstrong\u003eC. \u003c/strong\u003ePCa samples.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/b7cd7e9110fa872efc61b8b7.png"},{"id":88648055,"identity":"9111974a-ee09-4098-bddf-de439ee2ce37","added_by":"auto","created_at":"2025-08-08 16:53:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":392007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurvalumab induced changes\u003c/strong\u003e \u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e culture of fresh bladder cancer samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Expression of PD-L1 on non-immune cells (CD45\u003csup\u003e-\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0218\u003c/strong\u003e). \u003cstrong\u003eB.\u003c/strong\u003e Expression of PD-L1 by macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0078\u003c/strong\u003e).\u003cstrong\u003e C.\u003c/strong\u003e The proportion of ROS production by myeloid cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0039\u003c/strong\u003e). Results are presented with individual patient changes (left) and overall group changes (right).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/aba16753260ba901951c7204.png"},{"id":88649798,"identity":"694efb53-bf35-404a-be0b-8568c6705604","added_by":"auto","created_at":"2025-08-08 17:09:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":236578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurvalumab decreased PD-L1 expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e culture of fresh prostate cancer samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Expression of PD-L1 on non-immune cells (CD45\u003csup\u003e-\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0033\u003c/strong\u003e). \u003cstrong\u003eB.\u003c/strong\u003e Expression of PD-L1 by immune cells (CD45\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.033\u003c/strong\u003e).\u003cstrong\u003e \u003c/strong\u003eResults are presented with individual patient changes (left) and overall group changes (right).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/c60dfeca022aaf9a53d180c0.png"},{"id":88649235,"identity":"31bed6c5-9b4b-4bc2-a510-c15783f8a37f","added_by":"auto","created_at":"2025-08-08 17:01:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":419778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurvalumab induced changes\u003c/strong\u003e \u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e culture of fresh prostate cancer samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e ROS production by macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0431\u003c/strong\u003e). \u003cstrong\u003eB.\u003c/strong\u003e Phagocytosis capacity by immune cells (CD45\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0438\u003c/strong\u003e).\u003cstrong\u003e C.\u003c/strong\u003e Phagocytosis capacity by macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells) significantly decreased following durvalumab treatment (\u003cstrong\u003e*p=0.0211\u003c/strong\u003e). Results are presented with individual patient changes (left) and overall group changes (right).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/afe7a88d2ce116176312deec.png"},{"id":94989304,"identity":"e5461973-a798-4344-a09f-870cb1b1a76d","added_by":"auto","created_at":"2025-11-03 07:12:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2460680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/5c1967d9-3f9d-495d-bab7-6560e45e2a56.pdf"},{"id":88648054,"identity":"352c9584-645f-43b8-ba30-1115bd84c253","added_by":"auto","created_at":"2025-08-08 16:53:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":377940,"visible":true,"origin":"","legend":"","description":"","filename":"20250721SupplementaryData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7180379/v1/31af11ccc8baa8c1397dd088.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ex vivo culture to evaluate dynamic changes in the tumor microenvironment following durvalumab treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCheckpoint inhibition immunotherapy represents a significant paradigm shift in the treatment of many cancers, including bladder cancer (BCa). However, for others, it remains largely ineffective, such as in prostate cancer (PCa). These differences may be partly explained by genomic factors, with BCa recognized to have a very high tumor mutational burden (TMB) in contradistinction to PCa\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Up to 55% of the variation in efficacy of PD-1 inhibition has been estimated to be attributable to TMB\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Thus, other factors within the tumor microenvironment (TME) are significant contributors to influence response rates to immune checkpoint blockade. Understanding these differences and the heterogeneity between cancers represents an opportunity to identify immune resistance mechanisms which may be targeted to improve the proportion of responders.\u003c/p\u003e\u003cp\u003eThe TME is made of heterogenous cancer cells, stromal cells and immune cells, as part of a complex ecosystem\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The continuum of cancer immune surveillance, as well as its natural history and it’s receptivity to treatment, are defined by the spatial distribution and functional orientation of immune cells infiltrating the tumor\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Immune checkpoints such as PD-1/PD-L1 negatively regulate the immune system, are present on various cells including macrophages\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The PD-1/PD-L1 system may attenuate local T cell activation, proliferation, survival and cytotoxic secretion within cancer and decrease tissue damage\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Macrophages are often conceptually dichotomized as M1 or proinflammatory macrophages and M2 anti-inflammatory macrophages\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Prior studies suggest a predominance of immunosuppressive myeloid cells is associated with poorer responses to anti-PD-1 therapy\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDurvalumab is a fully human monoclonal antibody targeting PD-L1 from binding to its receptors PD-1 and CD80. IMFINZI (Durvalumab for injection) is currently approved for clinical use in urothelial carcinoma\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and muscle invasive bladder cancer\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e in Canada. A trial in muscle-invasive bladder cancer has shown event-free and overall survival benefits when combining durvalumab with chemotherapy\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Understanding the changes which occur in the human TME following treatment with durvalumab may provide insights to improve treatment strategies.\u003c/p\u003e\u003cp\u003eWe performed a prospective study using \u003cem\u003eex vivo\u003c/em\u003e culture of biopsies from prostates and bladders that were surgically removed to investigate how durvalumab influences the phenotype of macrophages within the respective cancerous organs. The use of \u003cem\u003eex vivo\u003c/em\u003e culture represents an attractive model in that it permits direct evaluation of cells within the TME\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Given the explanted nature precludes the infiltration of immune cells and our prior experience, we focus on changes in myeloid cells after 72h of treatment\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The objective was to evaluate phenotypic changes in myeloid cells within the microenvironment of bladder and prostate tumors after durvalumab treatment. We additionally investigate for relationships between immune myeloid cell phenotypes and patient and tumor characteristics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePatient recruitment.\u003c/b\u003e Institutional research ethics committee (REC) approval was obtained from the CHU de Québec-Université Laval (#2019–4181 and #2020–4547). Patients undergoing radical cystectomy or radical prostatectomy for urothelial carcinoma or prostatic adenocarcinoma, respectively, were targeted for this study. Informed consent was obtained from all patients who contributed fresh samples to our URO-1 institutional biobank (#2012 − 1002), and all methods complied with applicable ethical guidelines and regulations. Seventy patients were recruited for this study (35 bladders and 35 prostates).\u003c/p\u003e\u003cp\u003eFor BCa patients, the inclusion criteria were age ≥18 years, the ability to give informed consent, histologically confirmed urothelial carcinoma with a scheduled radical cystectomy, visibility of the tumor on pre-operative imaging or history of multiple tumors. Patients with or without pre-operative chemotherapy were included. The exclusion criteria for BCa patients were aborted surgery, inadequate tissue obtainable from the pathologist, known autoimmune disorders, prior anti-PD-1/PD-L1 or similar immune checkpoint inhibition within 2 years, receipt of high-dose steroids within 2 weeks of surgery and non-urothelial carcinoma histology.\u003c/p\u003e\u003cp\u003eFor PCa patients, the inclusion criteria were age ≥18 years, ability to give informed consent, histologically confirmed prostate adenocarcinoma and a scheduled radical prostatectomy. The exclusion criteria for PCa patients were aborted surgery, inadequate tissue obtainable from the pathologist, known autoimmune disorders, patients who have received anti-PD-1/PD-L1 or similar immune checkpoint inhibition within 2 years, receipt of high-dose steroids within 2 weeks of surgery, Gleason grade group 1 prostatic adenocarcinoma or non-adenocarcinoma histology.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBladder and prostate biopsy procurement.\u003c/b\u003e Biopsies for BCa were performed with 18G biopsies in the tumor area and a 5mm dermatological punch in the normal area. Biopsies for PCa were performed with 18G biopsies in the tumor area and in the normal area (1/3, 2/3 ratio) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eculture of biopsies.\u003c/b\u003e Technique was based on our previous work with PCa biopsies\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, with optimisation studies to select the optimal methods for bladder tissues. As previously described, bladder and prostate biopsies were first washed with HBSS Ca\u003csup\u003e2+\u003c/sup\u003e Mg\u003csup\u003e2+\u003c/sup\u003e (#311-513-CL, Wisent Bioproducts, St-Bruno, QC, Canada). Washed biopsies were then cut into two pieces; punches were cut in four pieces directly on plate with HBSS Ca\u003csup\u003e2+\u003c/sup\u003e Mg\u003csup\u003e2+\u003c/sup\u003e to keep tissues moist, with both pieces separated in a matched manner between isotype control (AstraZeneca, Cambridge, UK, 20µg/ml) and durvalumab (AstraZeneca, 20µg/ml) treatment for 72h. That is, a sample of normal adjacent tissue and of tumor sample were placed in each condition to balance comparisons. The biopsies were cultured in DMEM-F12 (#319-090-CL, Wisent Bioproducts) with 10% autologous serum obtained immediately prior to surgery, Glutamax (#35050061, ThermoFisher Scientific, Ottawa, ON, Canada, 1000mg/ml), HEPES (#15630080, Sigma-Aldrich, Saint-Louis, MO, 25mM) and Primocin (#ANT-PM-05, InvivoGen, San Diego, CA, 10ml/L) at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTissue dissociation for cytometry analysis.\u003c/b\u003e For BCa, biopsies were minced manually and using a GentleMACs dissociator (Miltenyi Biotec, San Diego, CA). Dissociated tissues were incubated with Collagenase III (#LS004182, Wisent Bioproducts, 10µl/10ml) for 30 min of agitation at 37°C followed by DNAse 1 (#260913, Sigma-Aldrich, 10µl/10ml) for 5 min of agitation at 37°C. The cellular suspension was filtered with a 70µm cell strainer. For PCa biopsies, the protocol was the same as the protocol for BCa except that Collagenase II (#17101015, Thermo Fisher, 1µl/ml) and DNAse 1 (Sigma-Aldrich, 1µl/ml) were added prior to overnight incubation. Accutase (#25-058-CL, Corning, NY) was also subsequently added to prostate biopsies, with a 70µm cell strainer used to remove clumps.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell staining and flow cytometry analysis.\u003c/b\u003e After dissociation, cells were incubated 10 min at room temperature with Fc block (#564219, BD Biosciences, Mississauga, Ontario, Canada) and then stained with an extracellular antibody panel for 30 min at 37°C, for 20 minutes for permeabilization and for 30 min at 4°C in the dark for intracellular antibodies. Antibodies used are detailed in Supplementary Table\u0026nbsp;1. Cell viability was assessed using the Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA). Stained cells were acquired on a LSR Fortessa cytometer (BD Immunocytometry Systems, San Jose, CA) and data collection was performed using BD FACS Diva software (BD Bioscience). Data analyses were performed using FlowJo software (v.10.7.1, FlowJo, LLC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical correlation analyses.\u003c/b\u003e Chart review identified pre-operative characteristics of each patient, including laboratory, pathological, and clinical data, similar to previous work\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The pre-prostatectomy CT scan distance between the prostate and pubic symphysis was used to quantify the periprostatic fat. We choose to use the minimal measured distance and dichotomized this by the median\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e into high and low periprostatic fat categories.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analyses.\u003c/b\u003e Descriptive statistics evaluated immune cell composition of biopsies according to patient and tumor characteristics. A paired T-test evaluated differences in the relative expression of cells markers between isotype control and durvalumab treatment conditions in bladder and prostate biopsies. As an exploratory study, no correction was performed for multiple testing. Analyses were performed using GraphPad Prism 9.5.1. For clinical characteristics of the BCa and PCa cohort, means are presented ± standard deviation unless otherwise indicated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical characteristics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical characteristics of the BCa cohort and the PCa cohort are summarized in Table 1. For BCa the mean age was 67.7 \u0026plusmn; 10 years, 15 patients received neoadjuvant chemotherapy, the mean patient weight was 74.5 \u0026plusmn; 14.2 kg and the mean body mass index (BMI) was 26.0 \u0026plusmn; 4.3. For PCa, the mean age was 66.7 \u0026plusmn; 6 years, the mean weight was 79.2 \u0026plusmn; 12.8 kg and the mean BMI was 26.4 \u0026plusmn; 3.8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;culture to evaluate changes in immune cells within the BCa microenvironment.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn bladder samples we observed no significant change in the proportion of live cells following 72h of \u003cem\u003eex vivo\u003c/em\u003e treatment between durvalumab and isotype control (Figure 1B). Durvalumab decreased the proportion of PD-L1 expressing non-immune cells (CD45\u003csup\u003e-\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells, Figure 2A) and macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells, Figure 2B) in bladder samples. Further, there was a significant increase of reactive oxygen species (ROS) production by myeloid cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e cells, Figure 2C) after durvalumab treatment. No other significant differences were observed among both macrophage and general immune cell markers. This non-significant change in markers expression is represented in tSNE analyses on macrophages of all BCa patients treated with isotype or durvalumab (Supplementary Figure 1A-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEx vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;culture to evaluate changes in immune cells within the PCa microenvironment.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly, among the prostate biopsies, we observed good cell viability after 72h of treatment (Figure 1C). Further, in the prostate biopsies, durvalumab once more decreased the expression of the immune checkpoint PD-L1 in non-immune cells (CD45\u003csup\u003e-\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells, Figure 3A) and in immune cells (CD45\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e cells, Figure 3B). Among the macrophage population (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e cells), we observed that expression of ROS increased following durvalumab treatment compared to isotype control (Figure 4A). We also observed a concomitant decrease in the proportion of phagocytic immune cells (CD45\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells, Figure 4B) and phagocytic macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells, Figure 4C). Furthermore, the mean fluorescence intensity (MFI) of CD45\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells and CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePhago\u003csup\u003e+\u003c/sup\u003e cells were also significantly lower (data not shown). No other significant differences were observed among both macrophage and general immune cell markers. Likewise, tSNE analyses demonstrate non-significant changes in markers expression in macrophages of all PCa samples (Supplementary figure 1C-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelation between the macrophage-associated markers and clinical characteristics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing our above results in the isotype-control group, we next assessed for correlation between macrophage-lineage immunosuppressive markers (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLA-DR\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ecells) and specific clinical characteristic of patients (Table 1). For BCa patients (Table 2), there were no significant correlations between the individual expression of selected cell cytometry profiles and selected clinical characteristics, except for a negative correlation between CD206\u003csup\u003e+\u003c/sup\u003e macrophage expression and the presence of lymphovascular invasion (LVI, r=-0.3428; *p=0.0438), and a negative correlation between the PD-L2 macrophage expression and patient weight (r=-0.3465, *p=0.0415) or BMI (r=-0.5028, *p=0.0021).\u003c/p\u003e\n\u003cp\u003eFor the same analysis in PCa patients (Table 3), we observed a positive correlation between the CD206\u003csup\u003e+\u003c/sup\u003e macrophage expression and the presence of intraductal carcinoma of the prostate (IDC, r=0.3646, *p=0.0313), between the PD-L1 macrophage expression in macrophages and the prostate tumor volume (PTV, r=0.335, *p=0.0492), and between macrophage ROS production and periprostatic fat (PF, r=0.461, *p=0.0354). In fact, ROS production by macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e cells) is significantly increased by durvalumab treatment in patients with low periprostatic fat whereas it is unchanged after durvalumab treatment in patients with high periprostatic fat (Supplementary figure 2). We also found a negative correlation between the abundance of macrophages and the presence of perineural invasion (PNI, r=-0.3677, *p=0.0456), between the CD163\u003csup\u003e+\u003c/sup\u003e macrophage expression and seminal vesicle invasion (SVI, r=-0.4337, *p=0.0092), and between the PD-L1 macrophage expression and PNI (r=-0.5001, *p=0.0049)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results using detailed flow cytometry demonstrate both the limits and utility of \u003cem\u003eex vivo\u003c/em\u003e culture to evaluate dynamic changes in myeloid cell markers. Our results demonstrate evidence of an expected decrease in PD-L1 expression following anti-PD-L1 treatment with durvalumab in both PCa and BCa patients. However, of the different immune phenotype markers evaluated, most changes were observed were only for functional markers: ROS production and phagocytosis activity. Moreover, we overall observed more significant changes in the prostate biopsies than the bladder samples. Notably, we showed that our culture methods did not affect cell viability after 72h, highlighting the utility for both other treatments and other tissue types.\u003c/p\u003e\u003cp\u003eThe observed decrease in PD-L1 expression on both immune and non-immune cells in prostate biopsies and in non-immune cells and macrophages in bladder samples suggests that durvalumab interacted with individual cells as expected during \u003cem\u003eex vivo\u003c/em\u003e treatment. With our cells washed significantly prior to flow cytometry, it is unlikely the decreases are related to durvalumab interfering with binding of antibody panel anti-PD-L1. We can infer that this decrease may have functional consequences. Prior research has suggested macrophage expression of PD-L1 contributes to active removal of T cells from the TME, suggesting a role for myeloid cells and PD-L1 inhibition in the trafficking of T cells in the TME\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We also observed a decrease of the PD-L1 expression in non-immune cells in both BCa and PCa samples following durvalumab treatment. It is a reasonable inference that most of these cells are cancer cells. Cancer cell expression of PD-L1 can directly suppressing the cytotoxic activity of PD1\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, though we did not evaluate T cells in our study.\u003c/p\u003e\u003cp\u003eOur novel finding of an increase in ROS production and a decrease of phagocytosis activity in macrophages in prostate biopsies suggests PD-L1 inhibition may induce a more favorable M1 macrophage phenotype within the prostate. These results also suggest a switch in macrophage phenotype (anti-inflammatory toward pro-inflammatory) in response to durvalumab.\u003c/p\u003e\u003cp\u003eIn our explorations of correlations between clinical characteristics and macrophages phenotypes we did not validate our prior findings with prostate biopsies\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, the positive correlation between the presence of IDC and the presence of M2 macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLA-DR\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e cells) did resemble our prior association of IDC with another (B7-H3\u003csup\u003e+\u003c/sup\u003e) immunosuppressive macrophage phenotype\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Interestingly, macrophage ROS production was positively correlated with the amount of periprostatic fat, suggesting a mechanism related to its link with more aggressive PCa\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In contrast to recent work in mouse PCa models suggesting a link between tumor-associated macrophage PD-1/PD-L1 expression and obesity-associated signaling\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we observed no correlation in human prostates between BMI and the presence of PD-L1\u003csup\u003e+\u003c/sup\u003e macrophages.\u003c/p\u003e\u003cp\u003eThe strengths of this study include the use of fresh patient samples to analyze the TME and the use of optimized flow cytometry to provide detailed information on cell and functional markers following durvalumab treatment. Nonetheless, as with all studies assessing patient samples, heterogeneity between the patients concerning their individual and tumor characteristics is confounding. Overall, our results suggest heterogeneity was greater among BCa patients. Similarly, while every effort was made to pair samples between treatments, tissue heterogeneity adds further limitations. Nonetheless, our methods yielded high cell viability and quality flow cytometry results. However, we only evaluated changes after 72h of treatment. The time frame for changes to the different markers evaluated is not known and this may explain why the more functional markers of ROS production and phagocytosis had the most detectable differences. Finally, our study was focused on macrophages, without data on other pertinent cells such as T cells.\u003c/p\u003e\u003cp\u003eIn summary, our detailed cytometric assessment of samples from BCa and PCa patients reveal the limits and utility of \u003cem\u003eex vivo\u003c/em\u003e treatment assessments of the TME. While observing expected changes in PD-L1 for both samples, relatively few overall changes were observed. Nonetheless, we notably observed an increase in myeloid ROS production in BCa and a similar increase in macrophage ROS production in PCa. Further, in PCa, a concomitant decrease in macrophage and immune cells phagocytosis was present. Together, our direct analyses of both tumor and adjacent normal tissue highlights the opportunities and challenges to capture overall trends in individual immune cells responses to treatment despite tissue and patient heterogeneity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Zohra Berrehail, Chlo\u0026eacute; Miranda, Maryke Venne and Genevi\u0026egrave;ve Tr\u0026eacute;panier for their contributions to this project.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor responsibilities:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: TG, PT\u003c/p\u003e\n\u003cp\u003eAcquisition of data: TG, HS, CM\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of data: TG, HS, PT\u003c/p\u003e\n\u003cp\u003eDrafting of the article: TG, HS\u003c/p\u003e\n\u003cp\u003eArticle revision for critically for important intellectual content: LL, VF, YF, FP, ML\u003c/p\u003e\n\u003cp\u003eStatistical analysis: TG, HS, PT\u003c/p\u003e\n\u003cp\u003eObtaining funding: PT\u003c/p\u003e\n\u003cp\u003eAdministrative, technical or material support: TG, CM\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: TG, PT\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential conflicts of interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePT reports research funding from AstraZeneca as well as personal fees as a consultant for Bayer, Knight, TerSera, and Abbvie. Other authors have no potential conflicts of interest to report.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by an externally sponsored research grant from AstraZeneca (ESR-19-20213, PT). \u0026nbsp;PT also is supported by a clinician-scientist award from Fonds de Recherche du Qu\u0026eacute;bec \u0026ndash; Sant\u0026eacute; (#354004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: TG, PTAcquisition of data: TG, HS, CMAnalysis and interpretation of data: TG, HS, PTDrafting of the article: TG, HSArticle revision for critically for important intellectual content: LL, VF, YF, FP, MLStatistical analysis: TG, HS, PTObtaining funding: PTAdministrative, technical or material support: TG, CM Supervision: TG, PT\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMartincorena, I. \u0026amp; Campbell, P. 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H.\u003cem\u003e et al.\u003c/em\u003e Periprostatic fat magnetic resonance imaging based radiomics nomogram for predicting biochemical recurrence-free survival in patients with non-metastatic prostate cancer after radical prostatectomy. \u003cem\u003eBMC Cancer\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1459, doi:10.1186/s12885-024-13207-4 (2024).\u003c/li\u003e\n\u003cli\u003eBader, J. E.\u003cem\u003e et al.\u003c/em\u003e Obesity induces PD-1 on macrophages to suppress anti-tumour immunity. \u003cem\u003eNature\u003c/em\u003e, doi:10.1038/s41586-024-07529-3 (2024).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eClinical\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003echaracteristics of patients with fresh prostate or bladder biopsy samples analyzed for myeloid cell markers by flow cytometry after a 72h of \u003cem\u003eex vivo\u003c/em\u003e culture. A total of 70 patients were evaluated (35 BCa patients and 35 PCa patients).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBladder cancer patients\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProstate cancer patients\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n=35)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e67.74 \u0026plusmn; 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e66.69 \u0026plusmn; 6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurgical Pathology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGleason Group Grade\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup Grade\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT Stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epTis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epTa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT3a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT3b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003epT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtraprostatic extension (EPE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntraductal carcinoma (IDC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN Stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurgical Margin status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean % Prostate Tumor Volume\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(min-max)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 189px;\"\u003e\n \u003cp\u003e17.46\u003c/p\u003e\n \u003cp\u003e(1-80)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Correlation between characteristics of BCa disease and macrophages associated markers in the macrophage population. Spearman\u0026rsquo;s rank correlation table show the correlation between specific characteristics of the BCa and the proportion of macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e cells) expressing M1 markers (CCR7, ROS expression), M2-associated markers (CD163, CD206) or immune checkpoints (B7H3, PD-L1 or PD-L2). Correlation coefficients (r\u003csub\u003es\u003c/sub\u003e) \u0026lt;-0.3 with significant p-values are indicated in bold. \u003cem\u003eNLR\u003c/em\u003e pre-operative neutrophil-to-lymphocyte ratio,\u003cem\u003e\u0026nbsp;LVI\u003c/em\u003e Lymphovascular invasion,\u003cem\u003e\u0026nbsp;BCG\u003c/em\u003e Bacillus Calmette-Guerin treatment,\u003cem\u003e\u0026nbsp;NAC\u0026nbsp;\u003c/em\u003eNeoadjuvant chemotherapy,\u003cem\u003e\u0026nbsp;PM\u003c/em\u003e Positive margin,\u003cem\u003e\u0026nbsp;W\u003c/em\u003e Weight and \u003cem\u003eBMI\u003c/em\u003e Body mass index.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"873\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eB7H3\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCCR7\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L2\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNLR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,09766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,009456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,06755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,02809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,204\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,008304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,07634\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,07839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,002393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,3428\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,03364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,2177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,04159\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,09964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,04501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,06345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,05452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,06961\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,0684\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1303\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,2491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,08728\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,2855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,1575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,2872\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,05635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,01759\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,01306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,08557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,08888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,08809\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eW\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,01397\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,02255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,06345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,06262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,3134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,09716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,3465\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1841\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,06914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,06666\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,1074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,3041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-0,06878\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,5028\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0,316\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e Correlation between characteristics of PCa disease and macrophages associated markers in the macrophage populations. Spearman\u0026rsquo;s rank correlation table show the correlation between specific characteristics of the PCa disease and the proportion of macrophages (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e cells) expressing M1 markers (CCR7, ROS expression), M2-associated markers (CD163, CD206) or immune checkpoints (B7H3, PD-L1 or PD-L2). Correlation coefficients (r\u003csub\u003es\u003c/sub\u003e) \u0026gt;0.3 or \u0026lt;-0.3 with significant p-values are highlighted in bold. \u003cem\u003eNLR\u003c/em\u003e Neutrophil-to-lymphocyte ratio, \u003cem\u003ePSA\u003c/em\u003e Prostate specific antigen, \u003cem\u003eEPE\u003c/em\u003e Extraprostatic extension, \u003cem\u003eSVI\u003c/em\u003e Seminal vesicle invasion, \u003cem\u003eIDC\u003c/em\u003e Intraductal carcinoma of the prostate, \u003cem\u003ePNI\u003c/em\u003e Perineural invasion,\u003cem\u003e\u0026nbsp;LVI\u003c/em\u003e Lymphovascular invasion,\u003cem\u003e\u0026nbsp;PTV\u0026nbsp;\u003c/em\u003eProstate tumor volume,\u003cem\u003e\u0026nbsp;PM\u003c/em\u003e Positive margin,\u003cem\u003e\u0026nbsp;W\u003c/em\u003e Weight, \u003cem\u003eBMI\u003c/em\u003e Body mass index and \u003cem\u003ePF\u003c/em\u003e Periprostatic fat.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eB7H3\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCCR7\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L1\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003ePD-L2\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eHLADR\u003csup\u003e+\u003c/sup\u003eROS\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNLR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0,7339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,01203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-0,004516\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0,1504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,2386\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e-0,2509\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e-0,07434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,05958\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePSA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,1618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,003981\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,06091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0,1841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e-0,05719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,002978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e-0,1145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,02607\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEPE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,2732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,1034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,1224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-0,2944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,2193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,0511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,07172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0,01983\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSVI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,2271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,2873\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,1715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,4337\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,1189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,1088\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0,08788\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e-0,1038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,1055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e0,1272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-0,1284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,3646\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,1373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0,1455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0,1915\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,3677\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-0,2358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10px;\"\u003e\n \u003cp\u003e-0,07885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e-0,2403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e-0,04449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0,5001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13px;\"\u003e\n 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cancer, prostate cancer, checkpoint inhibition, myeloid cells, tumor microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-7180379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7180379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile the tumor microenvironment (TME) is critical to the mechanism of action of anti-PD-1/PD-L1 checkpoint inhibitions, limitations exist to understand this using in vitro and in vivo models. With bladder (BCa) and prostate (PCa) cancer representing cancer with respectively strong and poor responses to PD-1/PD-L1 inhibition, we used ex vivo culture of fresh patient cancer tissue to better understand changes in the TME following durvalumab therapy, specifically the macrophage phenotype.\u003c/p\u003e\u003cp\u003eFresh prostate or bladder biopsies from 70 patients were cultured with durvalumab or isotype control. Flow cytometry assessed a panel of myeloid cell markers.\u003c/p\u003e\u003cp\u003eIn BCa samples, there was a significant difference, after durvalumab treatment, in PD-L1 expression on non-immune cells and macrophages and increased reactive oxygen species (ROS) production by myeloid cells in BCa. In PCa samples, there was a significant decrease in PD-L1 expression on non-immune and immune cells and increased ROS production by macrophages. Concomitantly, we observed a significant decrease in phagocytosis activity in immune cells and macrophages in prostate biopsies.\u003c/p\u003e\u003cp\u003eOur detailed cytometric analysis of ex vivo cultured BCa and PCa biopsies demonstrated changes in macrophage phenotype after durvalumab treatment. This study shows ex vivo culture can ascertain trends in tumor immune cell responses to immunotherapy.\u003c/p\u003e","manuscriptTitle":"Ex vivo culture to evaluate dynamic changes in the tumor microenvironment following durvalumab treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:52:56","doi":"10.21203/rs.3.rs-7180379/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a69b85ea-6740-41a1-aa6d-5c3851137131","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52542244,"name":"Biological sciences/Cancer"},{"id":52542245,"name":"Biological sciences/Immunology"},{"id":52542246,"name":"Health sciences/Oncology"},{"id":52542247,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2025-11-03T06:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-08 16:52:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7180379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7180379","identity":"rs-7180379","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}