Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response

Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response | 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 Research Article Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response PARAMESWAR DALAI, Dhruvi Shah, Kinal Soni, Jigna Shah, Chirag Desai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3977928/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 Purpose Studies have reported that cellular metabolism at tumor-immune microenvironment (TiME) serve as a critical checkpoint and perturbs/supports anti-cancer immunity. Extracellular ATP (eATP) may mediate anti-cancer immune response however; its catabolism by ectonucleotidase generates immunosuppressive adenosine. Antagonist of ectonucleotidases: CD39 and CD73 have been explored as potential therapeutic. In the presented work we have tried to repurpose doxycycline for mitigating ATP metabolism with or without antagonist of ectonucleotidase. Methods eATP and adenosine level were quantified. The bone marrow-derived M1 and M2 polarized macrophages were maintained in tumor mimicking condition (TMC). Total or CD4 + Tcells were co-cultured with macrophages to understand the impact of doxycycline and antagonist of ectonucleotidase T cell/subset differentiation. Preclinical efficacy of doxycycline and ectonucleotidase antagonist and their synergy was scored in 4T1 breast carcinoma. Results Doxycycline manipulates macrophage polarization by decreasing the frequency CD206 + M2 macrophages that promoted CD4 + directed CD8 + T cell mediated tumor cell lysis. Doxycycline alleviated the expression of CD39 and CD73, rescuing ATP catabolism. Doxycycline delayed tumor growth by enhancing F4/80 + CD86 + M1 macrophages and subsequently anti-tumor Tbet + CD4 + T-cells, attenuating the frequency of FOXP3 + regulatory T cells which was cooperatively supported by ARL67156 and AMPCP (CD39 and CD73 antagonist). Doxycycline promoted CD8 + T cell mediated cytotoxicity which was synergistically enhanced with ARL67156 and AMPCP ensuring a possibility of using doxycycline alone or in combination with antagonist of ectonucleotidase. Conclusion Presented data indicate a prospective usage of doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB. Doxycycline ectonucleotidase tumor microenvironment macrophage polarization T cells CD39 CD73 ARL67156 and AMPCP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Tumor-immune interactions in tumor microenvironment (TME) are important paradigm for depicting the fate of tumor growth and progression [ 1 ][ 2 ][ 3 ].Recently, therapeutics targeting immune checkpoint inhibition in TME is highly studied for their potential role in combating cancer progression. Although, immune checkpoint therapy is beneficial in some patients, most of cancer patients fail to respond to this [ 4 ][ 5 ].Hence, there is a dire need for a novel immunotherapeutic target to potentiate the therapeutic approach for anti-cancer treatments. Macrophage plasticity is one of the important mechanisms evolved in host to maintain homeostatic balance of immune response [ 6 ]. In the presence of external stimulus, macrophages are skewed into two major phenotypes: I.) Classically activated CD86 high iNOS + inflammatory (M1) macrophages and ii.) Alternatively activated CD206 high Arginase-1 + anti-inflammatory (M2) macrophages. Classically activated macrophages are critical in clearance of infection, while alternatively activated macrophages promote tissue repair, enhance angiogenesis and maintains homeostasis [ 7 ][ 8 ].Tumor-immune interactions play a critical role in macrophage recruitment and polarization. In TME, various factors contribute to promote the generation of immunosuppressive macrophages termed as Tumor associated macrophages (TAMs). [ 9 ][ 10 ] TAMs contribute to pro-tumorigenic outcome by enhancing angiogenesis, metastasis and immune suppression [ 11 ][ 12 ]. Cellular metabolism and metabolite availability in TME plays a vital role in shaping the outcome of immune and tumor cells [ 13 ].The metabolic adaptation by tumor and immune cells in TME highlights the importance of mitochondrial functions and ATP metabolism[ 14 ][ 15 ]. Antibiotics have garnered a great attention owing to their potential of affecting host mitochondrial functions. Cytotoxic chemotherapeutic potential of doxycycline is well established in breast cancer [ 16 ].Reports have indicated that doxycycline mediates dysregulation of mitochondrial membrane potential to induce tumor cell apoptosis. Apoptotic cell death is highly related with the increase in ATP levels [ 17 ]. In TME, extracellular ATP via P2X7R activation initiates a cascade of events ultimately leading to immune surveillance by cytotoxic T cell activation [ 18 ]. The ATP/adenosine levels are tightly regulated by membrane-bound ectonucleotidase. The hydrolysis of ATP to adenosine is catalysed by CD39 (ectonucleoside triphosphate diphosphohydrolase-1) and CD73 (ecto-5′-nucleotidase) enzymes. CD39 converts ATP/ADP to AMP that is further converted to adenosine by CD73. The elevated levels of adenosine are correlated with poor cancer prognosis [ 19 ]. Adenosine mediates immune suppression by enhancing macrophage polarization to TAMs [ 20 ]and proliferation of regulatory T cells and MDSC in TME [ 21 ],[ 22 ]. Apart from immune suppression, it promotes tumor cell proliferation and metastasis by catabolic energy production [ 21 ]. Additionally, TAMs also exhibit high expression of ectonucleotidase that in turn consume ATP to generate adenosine, maintaining immunosuppression in TME [ 23 ][ 24 ]. In TME, CD39 and CD73 ectonucleotidase are expressed not only on cancer cells but also on various immune cells such as regulatory T cells, tumor associated macrophages, tolerogenic DCs that contribute to immune regulation [ 25 ][ 26 ][ 27 ],the inhibition of these ectonucleotidase has shown a reduced tumor growth in different mouse models[ 26 ][ 28 ]. The combinational immunotherapy targeting metabolic checkpoint (ectonucleotidase) and immune checkpoint (PDL1-PD1) have shown a promising reduction in tumor growth of colon cancer model [ 29 ][ 30 ] [ 31 ]. In this manuscript, we have demonstrated that doxycycline enhanced anti-tumor immunity by inhibiting TAMs; promoting extracellular ATP release and partially suppressing the expression of ectonucleotidase on tumor and infiltrated immune cells in triple negative breast cancer model. Our study has revealed that repositioning doxycycline alone or in combination with antagonist of ectonucleotidase. Significantly reduced the tumor growth and promoted immune surveillance in TME. Thus, we report that doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB. 2. Materials and Methods 2.1 Reagents: All the recombinant proteins and antibodies used in the study are given in ( Supplementary table 1 and 2) with details of dilution used, clone and brand. 2.2 Cells lines: 4T1 cells were provided by Dr. Abhijit De (ACTREC Mumbai). The cells were cultured and maintained in RPMI supplemented with 10% FBS, 1x Penicillin-Streptomycin-Neomycin, at 37°C in a humidified incubator with 5%CO 2 . Raw264.7 macrophages were cultured and maintained in DMEM supplemented with 10% FBS, 1%Pencillin-Streptomycin-Neomycin, at 37°C in a humidified incubator with 5% CO 2 . 2.3 Bone marrow derived macrophage (BMDMs): BMDMs were generated following an already established lab protocol [ 32 ][ 33 ].Briefly, bone marrow cells were collected from femurs of BALB/c mice and cultured in RMPI-1650 medium supplemented with 10% FBS, 1x sodium pyruvate and HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) buffer. Cells were cultured in recombinant proteins M-CSF (20ng mL − 1 ) for macrophage generation (M 0 ). For polarization the cocktail of GM-CSF (20ng mL − 1 ) and IFNγ (10ng mL − 1 ) was used for M 1 macrophages while M-CSF (20ng mL − 1 ), IL-4(10ng mL − 1 ) and IL-10(10ng mL − 1 ) for M 2 macrophages. The cells were plat Extracellular ATP and Adenosine level were quantified by the help of manufacturing Kit. The bone marrow derives macrophages BMDMs was culture with tumor mimicking condition (TMC) for 3 days. The TCM-induced skewing macrophages to M2-like phenotype were confirmed by flow cytometry and ELISA. 2.4 Preparation of tumor mimicking conditioned media and cancer cell lysate: Crude soluble antigen (CSA) was prepared using freeze-thaw methodology as explained in [ 32 ].Confluent 4T1 cells were freeze-thawed for 7 repetitive cycles of -80°C/37°C for 10 min each and centrifuged at 10,000 rpm for 15 minutes. The protein concentration was quantified using Bradford assay. The culture supernatant was harvested from 95% confluent 4T1 cells maintained in RMPI containing 2% FBS in last 24 hrs.Tumor mimicking conditioned media (TMC) was prepared by mixing 10µg mL − 1 of CSA to 200µl of culture supernatant. These concentrations were maintained throughout the study and the TMC was used with fresh medium at 50:50 V/V to ensure nutritional availability [ 34 ]. 2.5 T cells isolation and macrophages -T cells co-culture: T cells isolation Spleen from the naive and tumor bearing mice was collected.T cells purified from the spleen after lysing RBCs using Geiss reagent (Sigma-Aldrich,USA).Total T cells were purified by passing through nylon wool.CD4 + T cells were purified from RBC-lysed splenocytes using an untouched CD4 + T cells enrichment kit(eBioscience)using manufacture protocol.CD8 + T cells were purified from RBC-lysed splenocytes using an untouched CD8 + T cells enrichment kit (eBioscience) using manufacturing protocol. [ 33 ][ 35 ]. Single cells suspension from tumor infiltrating lymph nodes (TILs) were prepared by mincing the lymph nodes between frosted end slides and the purified CD3 cells were used flow cytometric analysis of surface and intracellular marker of T cells. Macrophages -T cells co-culture : For macrophage and T-cells co-culture assays was done using the lab-established protocol [ 33 ].Briefly, BMDMs was generated in a 96 well plate following lab protocol [ 32 ]. After the generation of BMDMs treated with TMC in the absence/presence of doxycycline (20µg mL − 1 ) for 24 hr. Naive total T-cells/CD4 + T cells were added to the culture at a ratio of 1:10. Naive T cells without any stimulation served as a negative control. T cells were stimulated with CD3 and CD28 activating antibodies to analyse non-specific activation. After 3 days of co-culture in 96 well plates, IL-12, IFN-γ, IL-10 and TGF-β were quantified from the supernatant by ELISA. Simultaneously in a replicate experiment Brefildin-A was added to co-culture after 6hrs and incubated for 48hrs to analyse intracellular marker by flowcytometry or RT-PCR. The proliferation of T cells was measured either by MTT assays or CFSE proliferation assays. 2.6 Quantitative real time (RT)-PCR Gene expression was carried out by stabilised lab protocol [ 35 ].Briefly, the total RNA was extracted using TRIzol method and quantified. 2µg of RNA was used for cDNA synthesis by iScript cDNA synthesis kit (Bio-Rad USA).β-Actin was amplified from each sample to ensure equal cDNA input.100 ng of cDNA was used for amplification of mentioned genes in triplicates using gene-specific primers (supplementary table 3). Using power SYBR green 2X Master Mix in applied Bio systems) Step one plus Thermal cycler for 40 cycles and analysed with SDS 2.4 software (Applied Bio systems).Results normalized according to the expression levels of GAPDH mRNA. 2.7 Western Blot: Protein expression was analysed by western blotting [ 33 ]. Briefly Radio immuno precipitation Assay (RIPA) buffer Supplemented with Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor (PI) acceding to the manufacture’s recommended concentration was used to lysate cells. Each sample was normalized to 50 µg/30µl protein was resolved on an SDS-PAGE gel and electro-blotted onto a PVDF membrane. The membrane was blocked with 5% non-fat milk or 2% BSA in TBS for 2 h at RT. The membrane was probed overnight at 4˚c with following primary antibodies: sheep anti-mouse Arginase-1, Rabbit anti-mouse iNOS and GAPDH at (1:1000) dilutions. GAPDH was used as an internal control. Later, the membrane was probed with respective HRP- conjugated secondary antibody (1:5000) for 1 h at RT followed by the detection with ECL kit. 2.8 Flowcytometry analysis: Cells were collected and stained for flow cytometry analysis. For In vitro experiment we used BMDMs for macrophage polarization experiment. BMDMs polarized in to M 0 , M 1 and M 2 macrophages [ 32 , 33 ]and treated with TMC for 24 h followed by doxycycline (20µg/ml) treatment for 24hrs and surface protein was analysed by flow cytometry. The cell were analysed for expression of: CD11b-PE, CD86-FITC and CD206-APC. Details dilution and brand are mentioned in ( supplementary table 1 ). For In vivo experiment we used Single cell suspensions from tumor infiltrating lymph nodes and tumor tissue, for intracellular staining, Brefeldin-A solution in DMSO had been added 12hrs before collection at a concentration of 1µg/ml. Positive control T cells were activated with CD28/3. Staining with CD44, CD39, CD73, CD11b, F4/80, CD86, CD206, CD3, CD4, CD8, FOXP3, Tbet, IFN-γ, IL-10, TGF-β (250x dilution each) was done using lab optimizing protocol [ 34 ]. Details dilution and brand are mentioned in ( supplementary table 1 ). Cells were blocked with Fc block CD16/32 antibody for 30 min before staining as a background staining control. Everything was done on ice. The acquisition was done on Backman Coulter .Cytoflex research flow cytometer. Data were analyzed using CytExpert 2.4 from Beckman Coulter and FlowJo_V10. To quantify the cells recruited, equally weighed tissue was minced in the PBS and cells were collected in equal volumes of PBS. 2.9 ELISA : BMDM cells were seeded at a density of 10 5 cells/well in triplicates in 96-well plate and were treated was indicated. After24hrs cell free supernatants were analysed for cytokines by ELISA. 96-well plate were coated with the capture antibody in coating buffer (Carbonate buffer PH 9.1) overnight at 4˚C.The wells were washed with washing buffer (PBST with 0.05% Tween)for 5 times and block with blocking buffer (2.5% FBS in washing buffer).The collected cell free supernatants were added to the wells and incubated overnight at 4˚C.Later the wells were washed with buffer for 5 times and incubated with detection antibody (Biotin-labelled) in blocking buffer for 2hrs.After washing the wells were incubated with Streptavidin-HRP for 30 min in dark; following its incubation with TMB substrate for 15 min stop solution (2N orthophosphoric acid) was added to wells and reading were taken at 450nm.All the ELISA experiments were done in triplicates and O.D was used to deduced concentration of cytokines using standard graph[ 32 ][ 33 ] Details dilution and brand are mentioned in ( supplementary table 1 ). 2.10 ATP Determination Assays: 4T1 or BMDM cells were seeded into a 96-well plate at a density of 10,000 cells per well in 10% FBS containing media. The cells were treated with doxycycline 20µg/ml, in the presence of the Real Time-Glo™ Extracellular ATP Assay Reagent (Promega, GA5010-1KT)[ 36 ].Luminescence data was collected every 6 hours using a BMG POLAR star® plate reader 2.11 Adenosine Assays: Adenosine was determined using culture supernatant of 4T1, BMDM and T cells samples by Adenosine Quantification Assay Kit (Sigma, MAK433-1KT) [ 37 ].According to the manufacturing recommendations, we have used 20µL of frozen plasma samples to measure adenosine concentration. The plate was read on spectraMaxM2 Spectrofluorometer. The fluorescent product was excited at 535 nm and detected at 587 nm. 2.12 Cytotoxic T-cell assay : For CTL assay, in order to track proliferation of the 4T1 cells, 4T1 cells were stained with CFSE cell division tracker kit (Biolegend Cat#423801) according to the manufacturer’s protocol, Briefly,4T1 resuspended in PBS 10 6 cells per mL were stained with 2µl of 10mM CFSE per 10 6 cells to yield a final CFSE concentration of 1µM.4T1 cells were incubated at 37˚ C for 15 min, centrifuged at 500g for 8min,resuspended in RPMI to neutralize the Dye, and incubated at 37˚C for 30 min. Cells were centrifuged again, reseupended at 10 6 cells per mL, and stored at 37˚C until plating with the total T-cells. Later total T-cells were purified from splenocytes of different experimental groups using nylon wool column. Total T-cells were co-cultured with polarized macrophage as mentioned earlier.[ 32 ][ 33 ]. Later the primed T cells, harvested from each experimental group were plated with CFSE stained 4T1 at ratios of 1:100 for 5 days. These cells were centrifuged, the supernatants were stored at -20˚C for analysis of cytokines levels using ELISA and cells were stained with CD44-APC.The cytolytic activity of Tcells against 4T1 cells were analyzs by CFSE proliferation assay. 2.13 4T1 Tumor model A syngenic orthotropic mouse breast cancer model was established using 4T1 cells as previously reported [ 38 ] [ 39 ][ 40 ]Briefly, 4T1 cells (5*10 5 in PBS) were injected into BALB/C mice (n = 5) through subcutaneously mammary gland. After tumor was palpable, the mice were treated with, Doxycycline (50 mg kg − 1 of body weight), ARL67156 (2mg kg − 1 of body weight) and AMPCP (20mg kg − 1 of body weight) was injected intraperitoneally in 50µL PBS on days 10, 13, 16, 19, 22, 25, 28, and 31th days. The tumor size was measured every alternate day using Vernier callipers, and the volume was calculated using the formula (length*width*width)/2. The mice were sacrificed on day 34 of tumor inoculation and excised tumors were weighed. All experiments were conducted in accordance with the guidelines of the Institutional Animal Ethical Committee after obtaining a clearance at Nirma University, Ahmedabad India. Housing and handling of mice was in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) at Institute of Pharmacy Nirma University, Ahmedabad, India. Protocol No: IP/PCOL/FAC/32/2022/49 dated 20/09/2022. 2.14 Statistical tests: All the data were collected and analysed from minimum of three individual biological experiment replicates including animal experiments. One-way ANOVA with Turkey’s test was used to analyse the significance between the groups. Two-way ANOVA with Turkey’s multiple comparison tests was used to analyse the difference among subgroups and the difference among groups; and longitudinal values. Significance was denoted as p. (*** means p < 000.1; ** means p < 0.005; * means p < 0.01; n.s means not significant). In the figures * to denote the significance difference. These denotations were explained in the figure legend. 3. Results 3.1. Doxycycline reverts TMC-induced M2 macrophages to M1 phenotype to induce Th-1 response In our previous observations, we observed the anticancer potential of doxycycline on murine breast cancer cells (Supplementary Fig. 1A-I). Here, we wanted to check immunomodulatory potential of doxycycline on macrophage polarization. In order to generate TAMs in the in vitro conditions, we have cultured bone-marrow derived macrophages (BMDMs) in tumor mimicking conditions (TMC) as explained in materials and methods (2.3).We have observed that TMC-induced CD11b + macrophages showed high expression of M2 markers- CD206, Arginase-1 and low expression of M1 markers- CD86 and iNOS indicating that TMC skewed macrophages to an immunosuppressive M2 phenotype. Later; we polarized the macrophages to M1 and M2 phenotype. Macrophage polarization was confirmed with increased CD206 expression in M2 macrophages and increased CD86 expression in M1 macrophages respectively (Sup. Figure 2A).Polarized macrophages were later incubated in TMC in the presence/absence of doxycycline. We have observed that doxycycline treatment significantly decreased the frequency of CD11b + CD206 + M2 macrophages, while enhancingCD11b + CD86 + M1 macrophages in TMC-induced immunosuppressive microenvironment (Fig. 1 A, B). It was further confirmed by iNOS and ARG-1 expression. We observed that increase in iNOS expression and subsequent decrease in IL-10 and TGFβ cytokines in TMC induced M2-polarized macrophages moreover decreased ARG-1 expression and subsequent decrease in IL-10 and TGFβ cytokines in TMC induced M1 macrophages (Fig. 1 C-E). Thus, doxycycline enhanced the frequency of anti-tumor M1 macrophages in tumor mimicking conditions. It is reported that distinguished macrophage subset differentiates CD4 + T cells to a distinct T cell subset. In macrophage-CD4 + T cell co-culture assay, we observed that doxycycline treatment enhanced CD4 + T cell proliferation with subsequent increase in inflammatory T cell transcription factor Treg and cytokine (IFNγ) and decreased Foxp3 mRNA expression and immunosuppressive cytokines (IL-10, TGFβ) in both polarized macrophages (Fig. 1 F-I).These results gave us an indication that doxycycline via enhancing M1 macrophages modulated the adaptive immune response in TME. 3.2. Doxycycline delayed tumor growth by decreasing the frequency of CD206 + M2 macrophages in TME. The above results highlighted the importance of doxycycline on macrophage polarization in TME. Further, we wanted to extrapolate these observations in in vivo conditions using 4T1 induced carcinoma. We generated a syngeneic orthotropic breast cancer model using 4T1 breast cancer cells as explained in materials and methods ( 2.13 ). Doxycycline treatment (50mg/kg of BW) was given for 8 doses starting from Day 10 of tumor injection, once the tumor was palpable. The mice were sacrificed on Day 34 and tumor tissues from control and doxycycline treatment group were excised (Fig. 2 A).The weight and volume of tumor from both the groups were measured. The doxycycline group showed a significant decrease in tumor weight, volume and percentage of tumor regression compared to the control (Fig. 2 B-E). In order to understand the in vivo efficacy of doxycycline on macrophage polarization, we have checked the frequency of M1 (CD11b + CD86 + ) and M2 (CD11b + CD206 + ) macrophages in tumor tissue of control and treatment group. We have observed that tumor control had increased number of CD11b + CD206 + M2 macrophages compared to CD11b + CD86 + M1 macrophages (Fig. 2 F). It was evident with increased release of IL-6, IL-10 and TGF-β cytokines (Fig. 2 G). On the contrary, doxycycline treatment group showed decreased frequency of CD11b + CD206 + macrophages in the tumor tissue, along with increase in TNFα, IFNγ, and IL-12 (Fig. 2 F, G). These results were in accordance with the above obtained in vitro results. Later, we checked the frequency of CD4 + and CD8 + T cells from tumor infiltrating lymph nodes. Surprisingly, we observed that the tumor control had high number of CD4 + T cells compared to CD8 + T cells, while in doxycycline treatment group; the frequency of CD8 + T cells is significantly increased from 10.3–23.4%, quite crucial for promoting anti-tumor immunity. We also checked Cytokine level of lymph node supernatant (Fig. 2 J).Since, we observed the increased frequency of CD8 + T cells in doxycycline treat group, we checked for anti-tumor cytotoxic potential by CTL assay. In CTL assay, the activated T cells isolated from tumor infiltrating lymph nodes were co-cultured with 4T1 breast cancer cells for 5 days and were checked for 4T1 proliferation. We observed an increased CTL activity of doxycycline group with decreased proliferation of 4T1 cells and increased release of IFNγ and TNFα (Fig. 2 K, L). Overall, this data suggested that doxycycline enhanced anti-tumor immunity by improving anti-tumor innate and adaptive immunity. 3.3. Doxycycline promotes ATP release from cancer cells and alters downstream CD39 and CD73 expression on cancer and immune cells. It is reported that the balance between ATP release and ATP consumption is disturbed in TME, leading to the accumulation of extracellular ATP in the TME [ 41 ]. The extracellular ATP is hydrolysed by ectonucleotidase CD39 and CD73, metabolising it to adenosine which further aids in enhancing immune suppression [ 26 ]. Thus, inhibiting adenosine in TME aids in reduction of immune suppression in TME. Doxycycline is known to exhibit anti-tumor effect by targeting mitochondrial biogenesis and oxidative phosphorylation [ 42 ]. So, in order to elucidate the underlying mechanism of immune modulation by doxycycline in TME, we have focused on ATP metabolism in TME. We have observed that in breast cancer cells, there was an increase in ATP concentration upon doxycycline treatment on 18 hrs which gradually decreased in 36 hrs (Fig: 3A). Simultaneously, we observed that there was no significant difference in adenosine concentration in control and doxycycline treated breast cancer cells till 18 hrs. Interestingly, after 18hrs, an increase in adenosine concentration was observed in control cells but not in doxycycline treated cells (Fig. 3 B).Similarly, the doxycycline treatment on TMC-induced macrophages also showed similar observations (Fig. 3Cand D), indicating that doxycycline manipulated ATP metabolism in TME to further understand its effect on ATP metabolism, we checked for downstream ectonucleotidase -CD39 and CD73 expression in a time-dependent manner. Interestingly, we observed that the expression of CD39 and CD73 was similar in untreated and doxycycline-treated cancer cells till 18 hrs. After 18 hrs, there was a decrease in the CD39 and CD73 expression in doxycycline treated cancer cells (Fig. 3 E, F).This result aligned with our above observation of decreased adenosine concentration upon doxycycline treatment, indicating that doxycycline decreased CD73 and CD39 expression. Later, we extrapolated our findings to TMC-induced immunosuppressive macrophages. We observed that TMC-induced CD11b + macrophages had high expression of CD39 and CD73, which was decreased on doxycycline treatment in a time dependent manner (Fig. 3 G, H). These results indicate that doxycycline decreased CD39 and CD73 expression in a time dependent manner that in turn reduces adenosine levels to a lesser extent in TME. Additionally, we have checked the expression of ectonucleotidase in polarized macrophages. It was observed that immunosuppressive M2 macrophages showed high expression of CD73 and CD39 compared to anti-tumor M1 macrophages (SF, 4E). The frequency of CD39 + and CD73 + macrophages was increased in M1 and M2 macrophages when cultured in TMC. Doxycycline attenuated the frequency of CD39 + CD73 + TMC-induced macrophages in both polarized macrophages (Fig. 3 I-L).Overall data suggested that TMC-induced macrophages depict an immunosuppressive M2 phenotype with high expression of CD39 and CD73, which are abrogated by doxycycline in TME. 3.4. Antagonist of ectonucleotidase enhances doxycycline mediated anti-tumor immune response. Even though doxycycline restricts adenosine generation in TME, there is still a scope to the improve tumor weight and volume. In order to enhance the immunotherapeutic efficacy of doxycycline in context to ATP metabolism, we have tried to investigate the effect of doxycycline in combination with antagonist of CD39 (ARL67156) and CD73 (AMPCP). In murine breast cancer model, doxycycline (50mg/kg of BW), ARL67156 (2mg/kg of BW), AMPCP (20mg/kg of BW) were injected intraperitoneally in tumor bearing mice for 8 doses, every alternative day as explained in (Fig. 4 A). There was a significant reduction in the tumor weight and volume with no observed death in combination therapy of doxycycline with antagonist of ectonucleotidase (CD73 and CD39) in comparison to alone doxycycline/ CD39 and CD73 antagonists (Fig. 4 B-E).The inhibition of CD73 and CD39 by antagonists was confirmed with an increased in ATP concentration and decrease in adenosine levels in tumor tissue the reduction in adenosine levels was significantly high in the combination treatment of both antagonist along with doxycycline in comparison to doxycycline along with either antagonist (Fig. 4 F and G). In TME, it is reported that cancer cells and various immune cells such as regulatory T cells, TAMs exhibit high expression of ectonucleotidase (CD39&CD73) [ 43 ].So, we wanted to check the effect of antagonist and doxycycline treatment on ectonucleotidase in in-vivo conditions. In CD44 + breast cancer cells, we observed that the population of CD44 + CD39 + and CD44 + CD73 + cells was increased in tumor control, which on treatment with doxycycline and antagonist (ARL67156; AMPCP) was decreased to some extent. It was observed that combination of doxycycline along with antagonist limited CD73 + CD39 + population in the tumor tissue compared to alone treatments (Fig. 4 H&I). Similar results were obtained in tumor infiltrating immune cells (macrophages and T cells). The infiltrating immune cells of tumor group had high CD39 + CD73 + expressing cells which were abrogated in combination treatments (Fig. 4 J-M).Interestingly; we have observed that CD39 expressing cells were high in infiltrating myeloid cells, while CD73 expressing cells were high in lymphoid cells of tumor control. All these results indicated a synergy between doxycycline and CD73 CD39 antagonists, resulting in decreased tumor progression. 3.5. Combination treatment of doxycycline and antagonist (ARL67156, AMPCP) potentiates anti-tumor macrophages subset in TME. As in above observed results, the immunosuppressive M2 macrophages had an enhanced expression of CD73 and CD39 in comparison with M1 macrophages. So, we further wanted to check if this decrease in ectonucleotidase expression by combinational treatment skews tumor infiltrating macrophages to anti-tumor M1-phenotype. The tumor tissues from control and treatment groups were excised and stained for CD11b + CD86 + M1 macrophages and CD11b + CD206 + M2 macrophages. We observed that tumor group had significantly high frequency of M2 macrophages and low frequency of M1 macrophages. The tumor tissue of doxycycline treatment group had significantly enhanced frequency of M1 macrophages from 1.46–11.8%as compared to ARL67156 and AMPCP alone (Fig. 5 A). The population of M1 macrophages were improved in combination treatments. The combination treatment of doxycycline along with both antagonists potentiated anti-tumor macrophages to almost 27.7% in the tumor tissue (Fig. 5 A). We also observed increase the expression of iNOS from 5.05–60.8% and decrease the arginase-1 from 76.3–9.56% expression on combination treatment of doxycycline along with both antagonists. (Fig: 5B&C) The functional aspects of macrophages were confirmed by cytokine release. The excised tumor tissue from different groups were minced and checked for inflammatory and immunosuppressive cytokines. It was observed that supernatant of tumor control had high levels of IL-10 and TGF-β and low levels of IFN-γ, TNF-α. The levels of inflammatory cytokine (IFN-γ, TNF-α) were gradually increased from groups with alone treatment to combinational treatments (Fig. 5 D-G). This data were in coherence with the increase in M1 population in treatment groups indicating that the shift in cytokine milieu in TME is mediated by macrophage polarization. 3.6. Antagonists of ectonucleotidase synergizes with doxycycline to attenuate regulatory T cells in TME. The immunosurveillance by adaptive immune cells is essential to restrict tumor growth and development [ 44 ].Since we observed that doxycycline alone or in combination with both antagonists enhances innate immunity; we wanted to check its outcome on adaptive immunity. The total lymphocytes were isolated from tumor infiltrating lymph nodes of different experimental groups. The CD3 + CD4 + T cells were significantly increased in tumor control group than naïve from 26.7–70.9% (Fig. 6 A). Surprisingly, we observed that doxycycline along with antagonists of CD73 and CD39 in combinations had decreased the population of CD3 + CD4 + T cells from 70.9–26.6% as compare to along treatment (Fig. 6 A). The CD3 + CD4 + T cells subsets: T H -1, T H -2 and regulatory T cells in crucial in determining the outcome of tumor progression/regression [ 45 ]. Recruitment of regulatory T cells in TME is one of major mechanism adopted by tumor cells for immune evasion [ 45 ],[ 46 ]. Regulatory T cells are major contributors to extracellular adenosine-dependent immune suppression in TME [ 21 ].The lymph node of tumor control group showed low frequency of CD4 + Tbet + IFN-γ low secreting T H -1subsets(Fig. 6 B &C);and high frequency of CD4 + FOXP3 + IL-10 high + TGFβ high regulatory T cells (Fig. 6 D-F).In treatment groups, enhanced T H -1 population as evident with increase in CD4 + Tbet + IFNγ high T cell subsets which were further improved in combination treatment (Fig. 6 B&C). On the other hand, we have observed that treatments showed decrease in frequency of CD4 + FOXP3 + TGFβ high regulatory T cells which was improved in combination treatment (Fig. 6 D-F).It was further confirmed with decrease in released IL-10 and TGFβ cytokines (Fig. 6 I&J).These data were additionally confirmed with increase in IFNγ and TNFα analysed from the supernatants of tumor draining lymph node (Fig. 6 G&H). All these data indicate tumor group consist of high frequency of regulatory T cells while doxycycline and antagonists alone/in combination skew the ratio of pro-tumor immunity to anti-tumor immunity by enhancing Th1 phenotype. The combination treatment of both antagonists along with doxycycline exerts a better immune outcome than alone treatments/ in combination with either antagonist. 3.7. Combination treatment exerts an immune protective response by enhancing CD8 T cells mediated cytotoxicity The studies have reported a positive correlation between infiltrating CD3 + CD8 + T cells and enhanced survival of cancer patients [ 47 ], [ 48 ],[ 49 ]. In the Fig. 2 , we have observed that doxycycline enhanced the frequency of CD3 + CD8 + T cells rather than CD3 + CD4 + T cells in tumor bearing mice. As we observed a decrease in CD3 + CD4 + T cell population in treatment groups of tumor bearing mice, we checked the population of CD8 + T cells in treatment groups of tumor bearing mice. We observed that not only doxycycline but CD73 and CD39 antagonists also enhanced CD3 + CD8 + T cells population with subsequent IFN-γ (Fig. 7 A and B). The immunosuppressive factors in TME are reported to mediate CD3 + CD8 + T cell exhaustion with low cytotoxic activity. It is reported thatCD39 is a signature marker for CD3 + CD8 + T cell exhaustion [ 50 ].Interestingly, inCD3 + CD8 + T cells, we have observed a reduction in CD39 and CD73 expression in doxycycline alone or in combination with antagonists of tumor bearing mice compared to the tumor control (Fig. 7 C&D).The ultimate immune outcome in cancer regression is mediated by cytolytic activity of activated CD3 + CD8 + T cells. In order to confirm the functional aspect of CD8 + T cells. We checked for anti-tumor cytotoxic potential by CTL assay. In CTL assay, total T cells were isolated from lymph node of different experimental groups and co-cultured with 4T1 breast cancer cells. We have observed that doxycycline along with antagonist of CD39 and CD73 combinational group showed decreased viability of 4T1 cells that is from 89.5–32.0% moreover its increased cytokines level of IFN-γ and TNFα and decreased cytokine level of IL-10 and TGF-β (Fig. 7 E-I) Overall results indicated that doxycycline or antagonists of CD73 and CD39 alone skewed the immune response to antitumor immunity but when given in combination, mediated a better synergy and exerts an efficient innate and adaptive anti-tumor immune response, making it a promising immunotherapeutic strategy. 4. Discussion In this study, we have demonstrated the immune modulation potential of doxycycline in TME and the importance of doxycycline repositioning with metabolic checkpoint inhibition in TME. Our study highlights the usage of combinational therapy of doxycycline and antagonists of CD39 and CD73 to improve clinical outcomes in triple negative breast cancer (TNBC). Triple negative breast cancer is highly prevailing disease worldwide with no promising treatment modality [ 51 ][ 52 ][ 53 ][ 54 ]. Hence, identification of novel therapeutic approach that could potentially impact the disease is quite essential. Monocytes in tissue microenvironment differentiate to either inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Inflammatory macrophages are immune-stimulatory, important for clearance of infection, while M2 subset aids in tissue repair and maintaining homeostasis [ 55 ]. In TME, macrophages, on encounter with tumor-derived factors, polarize to M2-like subset termed as TAMs. An abundant infiltration of TAMs is highly correlated with the severity of the disease progression [ 56 ][ 57 ]. TAMs play a pivotal role in mediating immune evasion and are responsible for the failure of immunotherapeutic treatments. Thus, strategies targeting repolarization of TAMs to anti-tumor subset have received a great attention [ 58 ] [ 59 ] [ 60 ].Tetracyclines like doxycycline have been explored for their anti-cancer potential in several cancer models [ 61 ][ 62 ][ 63 ] [ 40 ]. It is reported to decrease the tumor growth and delay the reoccurrence of tumor in murine models [ 40 ]. Recently, the studies are focused on understanding the immune modulatory potential of doxycycline. In the model of choroidal neovascularization, doxycycline inhibits M2-polarization in dose-dependent manner [ 64 ][ 40 ]. In this study, we have investigated the immune modulatory potential of doxycycline on macrophage polarization in TME. We have observed that doxycycline treatment inhibited CD206 + TAMs in tumor tissue of TNBC. Additionally, we have observed that doxycycline treated mice showed reduced tumor growth and enhanced CD4 + and CD8 + T cell frequency compared to control mice. This finding suggests that doxycycline potentiated anti-cancer efficacy by enhancing host anti-tumor immune responses. One of the study has reported the important use of doxycycline in combination with chemotherapeutic drugs is because of its ability to target mitochondria [ 42 ]. The treatment with doxycycline induces mitochondrial dysregulation by altering membrane potential that ultimately leads to apoptotic cell death [ 65 ][ 16 ][ 66 ]. Upon apoptotic cell death, ATP is released in the extracellular microenvironment, affecting cellular metabolism in TME [ 67 ][ 27 ]. We have observed that treatment with doxycycline on TNBC promoted the release of ATP compared to control cells. In TME, the extracellular ATP is degraded to adenosine by ectonucleotidase (CD39 and CD73) [ 26 ][ 68 ]. The over expression of ectonucleotidase has been well documented in various cancer models. In TNBC patients, the ectonucleotidase CD39 and CD73 are highly expressed on tumor cells and infiltrating immune cells [ 26 ], [ 69 ] The generated adenosine further contributes to immune suppression by inhibiting antigen presentation, generation of TAMs and recruitment of regulatory T cells[ 70 ][ 21 ][ 71 ]. Numerous studies have shown that CD39-/- or CD73-/- mice inhibited tumor growth and experimental metastasis, suggesting that ATP/CD39-CD73/adenosine axis is important for immune suppression [ 24 ] [ 72 ]. Interestingly, we have observed that doxycycline treatment decreased adenosine levels on 4T1 cells and 4T1-induced immunosuppressive macrophages after 18 hrs of treatment (Fig. 3 B and D). So, we have explored the time-dependent expression of ectonucleotidase and effect of doxycycline on intrinsic CD39 and CD73 expression in tumor and infiltrating immune cells. We have observed that intrinsic expression of CD39 and CD73 in 4T1 cells increased after 18 hrs (SF.4 A-C). Additionally, we have observed doxycycline treatment partially abrogated the CD39 and CD73 expression on 4T1 and M2 macrophages (which had high expression compared to M1 macrophages). Thus, our data revealed that doxycycline exhibited immunotherapeutic efficacy by suppressing ectoenzyme mediated generation of TAMs and subsequent immune suppression in TME. In various cancer cell model, preclinical and clinical trials of small molecule inhibitors of ectonucleotidase CD39 and CD73 promoted anti-tumor immunity by stimulating APCs and restoring IFNγ secreting T cells [ 26 ][ 24 ][ 72 ]. Although doxycycline partially decreased the expression of CD39 and CD73, but absolute decrease in their expression would attenuate tumor growth and improve the overall survival. So, in this study, we tried to explore the combination effect of doxycycline along with the antagonists of CD73 (AMPCP) and CD39 (ARL67156). The combinational therapy of doxycycline and antagonists significantly decreased the tumor growth and enhanced the overall survival of tumor bearing mice. It also reduced the expression of CD39 and CD73 on tumor and infiltrating immune cells (macrophages and T cells), resulting in decreased adenosine levels in TME. As in observed results, the immunosuppressive M2 macrophages had an enhanced expression of CD73 and CD39 in comparison with M1 macrophages [ 73 ]. So, we further wanted to check if this decrease in ectoenzyme expression by combinational treatment skews tumor infiltrating macrophages to anti-tumor M1-phenotype Subsequently, we have observed an increase in the frequency of F4/80 + CD68 + M1 in the combinational therapy of doxycycline and antagonists compared to individual components. In TME, regulatory T cells consume ATP to produce adenosine that via A2AR/A2B receptor activation inhibits effector CD4 and CD8 T cells, generating T cell anergy [ 74 ][ 75 ].In addition to this, the generated adenosine aids in expansion of regulatory T cells by A2AR activation on regulatory T cells [ 74 ][ 76 ]Thus, adenosine maintains a positive feedback loop to establish immunosuppressive microenvironment [ 74 ]. The doxycycline alone or in combination with antagonist of ectonucleotidase therapy promotes the ratio of Foxp-3 + IL-10 + regulatory T cells/Tbet + IFN-γ + Th1 to Th1 phenotype shifting balance of T cells from immune evasion to immunosurveillance. Thus, our study provides strong evidence that repositioning doxycycline alone or in combination with antagonist of ectonucleotidase. Significantly reduced the tumor growth and promoted immune surveillance in TME. Thus, we report that doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB. Declarations Author contribution: PD designed and performed the experiments; analysed the data; prepared figures; drafted the manuscript and revisions. DS helped in animal experiments and analysed data. KS, KT helped in animal experiments. JS & CD extended animal facility, RAR conceived the study, designed the experiments and prepared and finalised the manuscript. Acknowledgement: Authors thank Dr. Abhijit de, ACTREC, Mumbai for providing TNBC-4T1 cell line. PD fellowship is supported by Indian Council of Medical Research (ICMR) and ScHemeOf Developing High quality research (SHODH); KS and SY fellowship were supported by Indian Council of Medical Research (ICMR); DS fellowship is supported by Lady Tata Memorial Trust (LTMT). This study was financially supported by Gujarat State Biotechnology Mission (GSBTM). Authors acknowledge Mansi Vaghela Hima Vora, Miloni Mehta and for their support in routine lab work. Conflict of Interest: There is no conflict of interest among the authors. Ethical Approval: We have declared that all experiments were conducted according to the guidelines of the Institutional Animal Ethical Committee after obtaining a clearance at Nirma University, Ahmedabad India. Protocol No: IP/PCOL/FAC/32/2022/49 . Funding: We have declared that this study was financially supported by the Gujarat State Biotechnology Mission (GSBTM), Department of Science & Technology and Government of Gujarat with Grant No. GSBTM/JD (R&D)/610/20-21/345. References Whiteside, T. The tumor microenvironment and its role in promoting tumor growth. Oncogene 27 , 5904–5912 (2008). https://doi.org/10.1038/onc.2008.271 Hinshaw, D. C., &Shevde, L. A. (2019). The Tumor Microenvironment Innately Modulates Cancer Progression. 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The Journal of biological chemistry , 292 (4), 1211–1217. https://doi.org/10.1074/jbc.C116.764043 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.pptx SupplementaryTable.docx GraphicalAbstract.pptx Supplementaryfigurelegends.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3977928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274969447,"identity":"0c8b0089-1703-4c75-8e4f-d99ebead8c5f","order_by":0,"name":"PARAMESWAR DALAI","email":"","orcid":"","institution":"Indian Institute of Advanced Research","correspondingAuthor":false,"prefix":"","firstName":"PARAMESWAR","middleName":"","lastName":"DALAI","suffix":""},{"id":274969448,"identity":"802c3c25-dd15-49de-9e1b-7fc021fdf725","order_by":1,"name":"Dhruvi Shah","email":"","orcid":"","institution":"Indian Institute of Advanced Research","correspondingAuthor":false,"prefix":"","firstName":"Dhruvi","middleName":"","lastName":"Shah","suffix":""},{"id":274969449,"identity":"6b38ae75-8be7-4acf-92ae-21ab4ac52173","order_by":2,"name":"Kinal Soni","email":"","orcid":"","institution":"Nirma University","correspondingAuthor":false,"prefix":"","firstName":"Kinal","middleName":"","lastName":"Soni","suffix":""},{"id":274969450,"identity":"3347af85-7dd6-4342-aad8-960b36ad7851","order_by":3,"name":"Jigna Shah","email":"","orcid":"","institution":"Nirma University","correspondingAuthor":false,"prefix":"","firstName":"Jigna","middleName":"","lastName":"Shah","suffix":""},{"id":274969451,"identity":"e4eec26c-16df-42c4-ae18-35db901c4fbb","order_by":4,"name":"Chirag Desai","email":"","orcid":"","institution":"Hemato-Oncology Clinic, Vedanta","correspondingAuthor":false,"prefix":"","firstName":"Chirag","middleName":"","lastName":"Desai","suffix":""},{"id":274969452,"identity":"bb82613b-7271-4b87-83f0-92e3a83e1b97","order_by":5,"name":"Kavya Thanki","email":"","orcid":"","institution":"Indian Institute of Advanced Research","correspondingAuthor":false,"prefix":"","firstName":"Kavya","middleName":"","lastName":"Thanki","suffix":""},{"id":274969453,"identity":"688ec88c-3368-4abc-925c-e568e4e7ed50","order_by":6,"name":"Reena Agrawal-Rajput","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYPACCwj1wcAGSDI2HiBCiwSYZJxRkAaiGojXwszz4TCYgVcLf//ah595aiTy+dl7D37gMThvt7b9MNCWGptonMbfeG4szXNMwnJmz7lkCQmD28nbziQCtRxLy23ApefGMQbpHDYJA4MbOQZA8nay2QGgFsaGwzi1yN84xvw755+Egf39N8Y/EgzOJZudf4hfi8H5Njbp3Dag+RI8ZhIHDA7Ymd0gYIvhDTY26799EgYSZ3LMLBsMkhPMbgBtScDjF7nzx5hvzvhmY8Dffsb49p8/dvZm59MfPvhQY4Pb+xIJqPxEsMoEDHVIgP8AKt8en+JRMApGwSgYmQAAeSViWqIVk48AAAAASUVORK5CYII=","orcid":"","institution":"Indian Institute of Advanced Research","correspondingAuthor":true,"prefix":"","firstName":"Reena","middleName":"","lastName":"Agrawal-Rajput","suffix":""}],"badges":[],"createdAt":"2024-02-22 07:31:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3977928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3977928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51729106,"identity":"db388a85-0473-4623-88e1-616849b270c7","added_by":"auto","created_at":"2024-02-28 04:03:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":548242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDoxycycline reverts TMC-induced M2 macrophages to M1 subsets to induced Th1 response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDM were polarized to M1 and M2 phenotype and treated with TMC in the presence of doxycycline (20μg/ml) for 24 hrs and analyzed for surface markers CD11b-PE, CD86-FITC and CD206-APC by flow cytometry (A).The gating strategy is explained in supplementary figure (SF.2A\u003cstrong\u003e).\u003c/strong\u003e The polarised M1 and M2 macrophages were treated with TMC in the presence of doxycycline for 6 hrs and mRNA expression of CD86, CD206, iNOS and ARG-1 was analysed using real-time qPCR and plotted as log fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e).GAPDH was used as control (B). The polarized M1 and M2 macrophages were treated with TMC in the presence/absence with doxycycline (20μg/ml) for 24hrs and analyzed for iNOS and Arginase-1 protein expression by western blot. GAPDH was used as loading control(C, D) (SF.2B).The supernatant of polarized macrophages were collected upon TMC and doxycycline (20μg/ml) treatment for 24 hrs and analyzed for secreted cytokines (TNFα, IL-12, IFNγ, IL-10 and TGFβ) by ELISA (E). Pictorial representation of Co-Culture between polarized macrophages M1 and M2 and total T-cells isolated from spleen of Naïve mice (F). Polarized macrophages were pre-treated with TCM and Doxycycline (20μg/ml) \u0026nbsp;for 24 hrs and co-cultured with CD4\u003csup\u003e+\u003c/sup\u003eT cells isolated from spleen of the naïve mice for 72 hrs and analysed for CD4\u003csup\u003e+\u003c/sup\u003eT cell proliferation by MTT assay(G).In a parallel experiment, after incubation for 48hrs, cells was analysed for Transcription factor (Tbet and FOXP3) and supernatant were analysed for secreted cytokines (IFNγ, TNFα, TGFβ and IL-10) were measured by RT-PCR and ELISA(H,I).Representative Figure-1 Group name mention as M1-Macrophages(Mφ)=M1 polarized macrophages, M2Macrophages(Mφ)=M2polarizedmacrophages, UT=Untreated, Dox=Doxycycline, TMC=Tumor mimicking conditions. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way\u0026nbsp; ANOVA with Turkey’s multiple comparison (***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/33f27bfd18c1d7ac362bccd6.png"},{"id":51729111,"identity":"830e9fe8-c0a3-4982-9a85-b350be2002a5","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":758353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDoxycycline delayed tumor growth by decreasing the frequency of CD206+ M2 macrophages in TME.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were injected subcutaneously in mammary fat pad using a tuberculin syringe in female BALB/c mice. Doxycycline (50 mg/kg) were injected intraperitoneally at 10\u003csup\u003eth\u003c/sup\u003e day and continued for 31 days at an interval of \u0026nbsp;3 days and mice were sacrificed at day 34 (n =5) (A). The volume, weight and size of tumors from control and treated group were measured and plotted (B-D).The % of tumor regression of Doxycycline treated mice’s and control mice’s was calculated (E).The excised Tumor tissue (Mixed population cells) are collected from tumor bearing mice as a control and doxycycline treatment group were stained for macrophage surface markers CD11b-PE, CD86-FITC and CD206-APC and analyzed by flowcytometry. The cells were then analyzed for CD11b, CD86 (M1 marker) and CD11b, CD206 (M2 marker).The gated CD11b positive cells were further gated to CD86 and CD206 positive cells respectively (F).Gating strategy explained in supplementary figure SF.2C.The excised tumor tissue from control and treatment groups was homogenized in the media and the supernatant was used to score cytokines (TNFα, IL-12, IFN-γ, IL-10, TGF β and IL-6) by ELISA (G).The excised Tumor infiltrating lymphocytes (TILs) from control and doxycycline treatment group were stained for CD3-PE Cy5.5, CD4-eFlour450 and CD8-APCCy7 expression was analyzed by flowcytometry and plotted as dot plots(H,I).Gating strategy explained in supplementary figure (SF:3A). The supernatant of tumor infiltrating lymphocytes (TILs) was used to analyze cytokines (IFNγ, TNFα, IL-10 and TGFβ) by ELISA (J). To determine the CTL activity, the purified total T cells from lymph node of different experimental groups were co-cultured with 4T1 breast cancer cells. Later, the cellular proliferation was assessed by CFSE proliferation assays by flow cytometry(k).Gating strategy explained in supplementary figure (SF.3D).In a parallel experiment, the supernatant was used to measure cytokines levels (TNFα, IFN-γ) by ELISA (L). Representative Figure Group name mention as Naïve=as a control, tumor=T, T+Dox=tumor+dox. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way ANOVA with Turkey’s multiple comparison (n=5***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/d1a5bc0cdd4fc871a5923c2d.png"},{"id":51729108,"identity":"8c68c4ba-818c-4dd1-8dad-c0417c0d78bf","added_by":"auto","created_at":"2024-02-28 04:03:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":660581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDoxycycline promotes ATP release from cancer cells and alters downstream CD39 and CD73 expression on cancer and immune cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were treated with doxycycline (20μg/ml) in the presence of real time-Glo extracellular ATP assay-reagent and measured for chemiluminescence for every 6hrs to determine extracellular ATP concentration (A). The supernatant were collected in time-dependent manner to determine adenosine levels using Adenosine quantification assay kit and plotted as Adenosine μM in sample (B). BMDMs cells were treated with TMC followed by doxycycline (20μg/ml) in the presence of real time-Glo extracellular ATP assay-reagent and measured for chemiluminescence for every 6hrs to determine extracellular ATP concentration(C). In a parallel experiment the supernatant were collected in time-dependent manner to determine adenosine levels using Adenosine quantification assay kit and plotted as µM in sample (D).The 4T1 were treated with doxycycline in time dependent manner and analyzed for CD44-APC Cy7, CD39-PE and CD73-FITC.The CD44\u003csup\u003e+\u003c/sup\u003e cells were further gated for CD73 and CD39 and plotted as histogram (E,F) Gating strategy explained in supplementary figure (SF:4A).The BMDMs were treated with doxycycline in the presence/absence of TMC in time dependent manner and analyzed for CD11b-APC,CD39-PE and CD73-FITC. The CD11b\u003csup\u003e+\u003c/sup\u003e cells were further gated for CD73 and CD39 and plotted as histogram (G, H) Gating strategy explained in supplementary figure (SF:4F). BMDM were polarized to M1 and M2 phenotype and treated with TMC in the presence/absence of doxycycline (20μg/ml) for 24 hrs and analyzed for surface markers CD11b-APC, CD39-PE and CD73-FITC by flow cytometry (I-L).The gating strategy is explained in supplementary figure (SF.5A\u003cstrong\u003e).\u003c/strong\u003eRepresentative Figure-3 Group name mention as UT=Untreated, Dox=Doxycycline.TMC=Tumor mimicking conditions, TMC+Dox=Tumor mimicking condition+Doxycycline. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by one-way\u0026nbsp; ANOVA with Turkey’s multiple comparison (***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/790451646e612338d1f12b01.png"},{"id":51729114,"identity":"ed216528-f310-4313-ae83-89bddbb9680f","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2054764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntagonist of ectonucleotidase enhances doxycycline mediated anti-tumor immune response:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were injected subcutaneously in mammary fat pad using tuberculin syringein female BALB/c mice. Doxycycline (50 mg/kg), ARL67156 (2mg/kg) and AMPCP (20mg/kg) were injected intraperitoneally at 10\u003csup\u003eth\u003c/sup\u003e day and continued for 31 days at an interval of 3 days and mice were sacrificed at day 34 (n =5) (A)The tumor size, tumor weight and tumor volume were determined and plotted (B,D). The survival curve of tumor bearing mice of different groups were calculated and plotted as percentage (E). The levels of eATP and adenosine were measured from supernatant of tumor tissue of various groups (F, G).The excised tumor tissue from different groups was stained for CD44-APC-Cy7, CD39-PE and CD73-FITC. The tumor cells were gated for CD44\u003csup\u003e+\u003c/sup\u003e vs CD39\u003csup\u003e+ \u003c/sup\u003ecells represent in contour plot (H). For CD44\u003csup\u003e+\u003c/sup\u003e cells and CD73\u003csup\u003e+ \u003c/sup\u003ecells in contour plot (I) Gating strategy explain in supplementary figure (SF:5B).For macrophages, the tumor tissues from tumor control and treated mice were collected and stained for CD11b-APC, CD39-PE, and CD73-FITC respectively. The tumor cells were gated for CD11b\u003csup\u003e+\u003c/sup\u003e cells and CD39\u003csup\u003e+ \u003c/sup\u003ecells in contour plot (J).For CD11b\u003csup\u003e+\u003c/sup\u003e cells and CD73\u003csup\u003e+ \u003c/sup\u003ecells in contour plot (K).Gating strategy explain in supplementary figure (SF: 5C).Tumor infiltrating lymph nodes (TILs) from control and treated mice were collected and stained for CD4-eFlour450, CD39-PE, CD73-FITC respectively. The lymph nodes cells were gated for CD4\u003csup\u003e+\u003c/sup\u003e cells and CD39\u003csup\u003e+ \u003c/sup\u003ecells in contour plot (L).For CD4\u003csup\u003e+\u003c/sup\u003e cells and CD73\u003csup\u003e+ \u003c/sup\u003ecells in contour plot (M).Gating strategy explain in supplementary figure(SF:6A) Representative Figure:4 Group name mention as Representative Figure:5 Group name mention as Tumor=T, T+Dox(D)=tumor+doxycycline,T+A-CD39(A39)=Tumor+ Antagonist of CD39,T+A-CD73=Tumor + Antagonist of CD73,T+D+A39=Tumor+ Doxycycline Antagonist of CD39,T+D+A73=Tumor+Doxycycline+Antagonist of CD73,T+D+A39+A73=Tumor+Doxycycline+Antagonist of CD39+antagonist of CD73. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way ANOVA with Turkey’s multiple comparison (n=5***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/601b0a47eda19e6b3eae3efd.png"},{"id":51729112,"identity":"ea53b809-b8f0-485b-88c1-d3e3d9b9cf8f","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":650861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination treatment of doxycycline and antagonist (ARL67156, AMPCP) potentiates anti-tumor macrophages subset in TME.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumor tissues from tumor control and treated mice were collected and stained for CD11b-PE, CD86-FITC, CD206-APC respectively analyzed by flow cytometry. The cells were then analyzed for CD11b, CD86 (M1 marker) and CD11b, CD206 (M2 marker).The gated CD11b positive cells were further gated to CD86 and CD206 positive cells respectively (A) Gating strategy explained in supplementary figure (SF.6B).The excised tumor tissue from the control and treatment group of tumor bearing mice was analysed for F4/80-FITC and iNOS APC for M1 Mφ and F4/80-FITC and ARG-1 PE for M2 Mφ (B, C). The gated F4/80 positive cells were further gated to iNOS and Arg-1 positive cells respectively. Gating strategy explained in supplementary figure (SF.7A).The supernatant from excised tumor tissue of different groups were analyzed for secreted cytokines (TNFα, IFNγ, IL-10 and TGF β) by ELISA (D-G). Representative Figure:5 Group name mention as Representative Figure:5 Group name mention as Tumor=T, T+Dox(D)=tumor+doxycycline,T+A-CD39(A39)=Tumor+ Antagonist of CD39,T+A-CD73=Tumor +Antagonist of CD73,T+D+A39=Tumor+ Doxycycline Antagonist of CD39,T+D+A73=Tumor+Doxycycline+Antagonist of CD73,T+D+A39+A73=Tumor+Doxycycline+Antagonist of CD39+antagonist of CD73. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way ANOVA with Turkey’s multiple comparison (n=5***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/948e3a4b84971c59515bedf0.png"},{"id":51729113,"identity":"eef37837-26e8-4219-aba5-6d4df34ab09c","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1326876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntagonists of \u0026nbsp;ectonucleotidase synergizes with doxycycline to attenuate regulatory T cells in TME.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe excised Tumor infiltrating lymphocytes (TILs) from the control and treated mice were collected and analysed for T cells markers: CD3-PE Cy5.5 and CD4-eFlour450 (A) Gating strategy explained in supplementary figure (SF:7B). In a parallel experiment, lymph node cells were stained for CD4-eFlour450, Tbet-PE, and IFNγ-APC. The lymph node cells were gated CD4\u003csup\u003e+\u003c/sup\u003e Tbet\u003csup\u003e+\u003c/sup\u003e cells represent as contour plot (B) CD4\u003csup\u003e+\u003c/sup\u003eIFN-γ cells represent as contour plot (C)Gating strategy explained in supplementary figure (SF:7B). In a parallel experiment, lymph node cells were stained for CD4-eFlour450, FOXP3-PE, TGF-β-PerCP-Cy 5.5 and IL-10-FITC.The CD4\u003csup\u003e+\u003c/sup\u003e cells were gated for FOXP3\u003csup\u003e+\u003c/sup\u003e cells represent as contour plot (D) Gating strategy explained in supplementary figure (SF:8A). CD4\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e\u0026nbsp; represent as contour plot (E) Gating strategy explained in supplementary figure (SF:8B).CD4\u003csup\u003e+\u003c/sup\u003eTGF-β cells\u0026nbsp; represent as contour plot (F).Gating statuary are same as (SF;8B) In a parallel experiment, the supernatant for Tumor infiltrating lymph node (TILs) \u0026nbsp;was used to analyze secreted cytokines (IFN-γ, TNF-α, TGF-β, IL-10) by ELISA (G-J). Representative Figure:6 Group name mention as Representative Figure:5 Group name mention as Tumor=T, T+Dox(D)=tumor+doxycycline,T+A-CD39(A39)=Tumor+ Antagonist of CD39,T+A-CD73=Tumor +Antagonist of CD73,T+D+A39=Tumor+ Doxycycline Antagonist of CD39,T+D+A73=Tumor+Doxycycline+Antagonist of CD73,T+D+A39+A73=Tumor+Doxycycline+Antagonist of CD39+antagonist of CD73. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way ANOVA with Turkey’s multiple comparison (n=5***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/2ba372ad211d4e783df451ae.png"},{"id":51729116,"identity":"a3d654cb-c3d9-4beb-b427-2ab52d7763ce","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1010748,"visible":true,"origin":"","legend":"\u003cp\u003eCombination treatment exerts an immune protective response by enhancing CD8 T cells mediated cytotoxicity:\u003c/p\u003e\n\u003cp\u003eThe excised tumor infiltrating lymph nodes from the control and treated mice were collected and analyzed for CD8 T cells markers: cells were stained with CD3-PE Cy5.5, CD8-APC Cy7 and IFNγ-APC. The gated CD3 positive cells were further gated to CD8 and IFN-γ positive cells repectively.The frequency of CD3 and CD8 T cells were analyzed from different treatment groups (A).Gating strategy explained in supplementary figure (SF:9A) The frequency of CD3 positive CD8 and \u0026nbsp;IFN-γ positive T cells \u0026nbsp;were analyzed from different treatment groups (B)In a parallel experiment tumor infiltrating lymph nodes from control and treated mice were collected and stained for CD8-APC Cy7, CD39-PE, and CD73-FITC respectively. The lymph nodes cells were gated for CD8+ cells and CD39+ cells in dot plot(C).For CD4+ cells and CD73+ cells in dot plot (D)Gating strategy same as supplementary figure (SF: 9B). To determine the CTL activity, the purified total T cells from lymph node of different experimental groups were co-cultured with 4T1 breast cancer cells. Later, the cellular proliferation was assessed by CFSE proliferation assays by flow cytometry(E).In a parallel experiment, the supernatant was used to measure cytokines levels (TNFα, IFN-γ,IL-10 and TGF-β) by ELISA (F-I) ). Representative Figure:7 Group name mention as Representative Figure:5 Group name mention as Tumor=T, T+Dox(D)=tumor+doxycycline,T+ACD39(A39)=Tumor+AntagonistofCD39, T+ACD73=Tumor+AntagonistofCD73,T+D+A39=Tumor+\u003c/p\u003e\n\u003cp\u003eDoxycycline+AntagonistofCD39, T+D+A73=Tumor+Doxycycline+AntagonistofCD73, T+D+A39+A73=Tumor+doxycycline+antagonist of CD39+antagonist of CD73. Data are of three experiments and represented as mean ± SEM. Statistical significance was determined by Two-way ANOVA with Turkey’s multiple comparison (n=5 ***p \u0026lt; 0.0001; **p \u0026lt; 0.001; *p \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/73b4801d6e9cca82973c5210.png"},{"id":53832374,"identity":"c1c7c51e-18c1-43b1-a27d-079b05557394","added_by":"auto","created_at":"2024-04-01 05:07:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3810399,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/1316f022-6d0c-451c-92a2-51db62ef093a.pdf"},{"id":51729110,"identity":"0ff67abb-e6b0-4096-8048-e46db02ffce7","added_by":"auto","created_at":"2024-02-28 04:03:09","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3680918,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/a010188769be9b37b6a2d478.pptx"},{"id":51729109,"identity":"e6746f8c-fefb-4109-ba55-dfe5ad781c29","added_by":"auto","created_at":"2024-02-28 04:03:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19298,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/925fa92521972cd72129c5f1.docx"},{"id":51729622,"identity":"b6e09e6d-6f63-44a6-9d57-787893ff5823","added_by":"auto","created_at":"2024-02-28 04:19:09","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1390346,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/97ba7026a417720a5faaf16e.pptx"},{"id":51729107,"identity":"dae44ca3-197f-483c-97dc-b548ad587bb1","added_by":"auto","created_at":"2024-02-28 04:03:08","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15779,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigurelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-3977928/v1/5f28cd25113abae9c8769421.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTumor-immune interactions in tumor microenvironment (TME) are important paradigm for depicting the fate of tumor growth and progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Recently, therapeutics targeting immune checkpoint inhibition in TME is highly studied for their potential role in combating cancer progression. Although, immune checkpoint therapy is beneficial in some patients, most of cancer patients fail to respond to this [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].Hence, there is a dire need for a novel immunotherapeutic target to potentiate the therapeutic approach for anti-cancer treatments. Macrophage plasticity is one of the important mechanisms evolved in host to maintain homeostatic balance of immune response [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the presence of external stimulus, macrophages are skewed into two major phenotypes: I.) Classically activated CD86 high iNOS\u0026thinsp;+\u0026thinsp;inflammatory (M1) macrophages and ii.) Alternatively activated CD206 high Arginase-1\u0026thinsp;+\u0026thinsp;anti-inflammatory (M2) macrophages. Classically activated macrophages are critical in clearance of infection, while alternatively activated macrophages promote tissue repair, enhance angiogenesis and maintains homeostasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].Tumor-immune interactions play a critical role in macrophage recruitment and polarization. In TME, various factors contribute to promote the generation of immunosuppressive macrophages termed as Tumor associated macrophages (TAMs). [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] TAMs contribute to pro-tumorigenic outcome by enhancing angiogenesis, metastasis and immune suppression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCellular metabolism and metabolite availability in TME plays a vital role in shaping the outcome of immune and tumor cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].The metabolic adaptation by tumor and immune cells in TME highlights the importance of mitochondrial functions and ATP metabolism[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Antibiotics have garnered a great attention owing to their potential of affecting host mitochondrial functions. Cytotoxic chemotherapeutic potential of doxycycline is well established in breast cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].Reports have indicated that doxycycline mediates dysregulation of mitochondrial membrane potential to induce tumor cell apoptosis. Apoptotic cell death is highly related with the increase in ATP levels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In TME, extracellular ATP via P2X7R activation initiates a cascade of events ultimately leading to immune surveillance by cytotoxic T cell activation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The ATP/adenosine levels are tightly regulated by membrane-bound ectonucleotidase. The hydrolysis of ATP to adenosine is catalysed by CD39 (ectonucleoside triphosphate diphosphohydrolase-1) and CD73 (ecto-5\u0026prime;-nucleotidase) enzymes. CD39 converts ATP/ADP to AMP that is further converted to adenosine by CD73. The elevated levels of adenosine are correlated with poor cancer prognosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Adenosine mediates immune suppression by enhancing macrophage polarization to TAMs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]and proliferation of regulatory T cells and MDSC in TME [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e],[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Apart from immune suppression, it promotes tumor cell proliferation and metastasis by catabolic energy production [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, TAMs also exhibit high expression of ectonucleotidase that in turn consume ATP to generate adenosine, maintaining immunosuppression in TME [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn TME, CD39 and CD73 ectonucleotidase are expressed not only on cancer cells but also on various immune cells such as regulatory T cells, tumor associated macrophages, tolerogenic DCs that contribute to immune regulation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e],the inhibition of these ectonucleotidase has shown a reduced tumor growth in different mouse models[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The combinational immunotherapy targeting metabolic checkpoint (ectonucleotidase) and immune checkpoint (PDL1-PD1) have shown a promising reduction in tumor growth of colon cancer model [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this manuscript, we have demonstrated that doxycycline enhanced anti-tumor immunity by inhibiting TAMs; promoting extracellular ATP release and partially suppressing the expression of ectonucleotidase on tumor and infiltrated immune cells in triple negative breast cancer model. Our study has revealed that repositioning doxycycline alone or in combination with antagonist of ectonucleotidase. Significantly reduced the tumor growth and promoted immune surveillance in TME. Thus, we report that doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents:\u003c/h2\u003e \u003cp\u003eAll the recombinant proteins and antibodies used in the study are given in (\u003cb\u003eSupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and 2)\u003c/b\u003e with details of dilution used, clone and brand.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cells lines:\u003c/h2\u003e \u003cp\u003e4T1 cells were provided by Dr. Abhijit De (ACTREC Mumbai). The cells were cultured and maintained in RPMI supplemented with 10% FBS, 1x Penicillin-Streptomycin-Neomycin, at 37\u0026deg;C in a humidified incubator with 5%CO\u003csub\u003e2\u003c/sub\u003e. Raw264.7 macrophages were cultured and maintained in DMEM supplemented with 10% FBS, 1%Pencillin-Streptomycin-Neomycin, at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bone marrow derived macrophage (BMDMs):\u003c/h2\u003e \u003cp\u003eBMDMs were generated following an already established lab protocol [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].Briefly, bone marrow cells were collected from femurs of BALB/c mice and cultured in RMPI-1650 medium supplemented with 10% FBS, 1x sodium pyruvate and HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) buffer. Cells were cultured in recombinant proteins M-CSF (20ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for macrophage generation (M\u003csub\u003e0\u003c/sub\u003e). For polarization the cocktail of GM-CSF (20ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and IFNγ (10ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used for M\u003csub\u003e1\u003c/sub\u003e macrophages while M-CSF (20ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), IL-4(10ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and IL-10(10ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for M\u003csub\u003e2\u003c/sub\u003e macrophages. The cells were plat Extracellular ATP and Adenosine level were quantified by the help of manufacturing Kit. The bone marrow derives macrophages BMDMs was culture with tumor mimicking condition (TMC) for 3 days. The TCM-induced skewing macrophages to M2-like phenotype were confirmed by flow cytometry and ELISA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of tumor mimicking conditioned media and cancer cell lysate:\u003c/h2\u003e \u003cp\u003eCrude soluble antigen (CSA) was prepared using freeze-thaw methodology as explained in [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].Confluent 4T1 cells were freeze-thawed for 7 repetitive cycles of -80\u0026deg;C/37\u0026deg;C for 10 min each and centrifuged at 10,000 rpm for 15 minutes. The protein concentration was quantified using Bradford assay. The culture supernatant was harvested from 95% confluent 4T1 cells maintained in RMPI containing 2% FBS in last 24 hrs.Tumor mimicking conditioned media (TMC) was prepared by mixing 10\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of CSA to 200\u0026micro;l of culture supernatant. These concentrations were maintained throughout the study and the TMC was used with fresh medium at 50:50 V/V to ensure nutritional availability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 T cells isolation and macrophages -T cells co-culture:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eT cells isolation\u003c/strong\u003e \u003cp\u003eSpleen from the naive and tumor bearing mice was collected.T cells purified from the spleen after lysing RBCs using Geiss reagent (Sigma-Aldrich,USA).Total T cells were purified by passing through nylon wool.CD4\u003csup\u003e+\u003c/sup\u003e T cells were purified from RBC-lysed splenocytes using an untouched CD4\u003csup\u003e+\u003c/sup\u003eT cells enrichment kit(eBioscience)using manufacture protocol.CD8\u003csup\u003e+\u003c/sup\u003e T cells were purified from RBC-lysed splenocytes using an untouched CD8\u003csup\u003e+\u003c/sup\u003e T cells enrichment kit (eBioscience) using manufacturing protocol. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e][\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/p\u003e \u003cp\u003eSingle cells suspension from tumor infiltrating lymph nodes (TILs) were prepared by mincing the lymph nodes between frosted end slides and the purified CD3 cells were used flow cytometric analysis of surface and intracellular marker of T cells.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eMacrophages -T cells co-culture\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eFor macrophage and T-cells co-culture assays was done using the lab-established protocol [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].Briefly, BMDMs was generated in a 96 well plate following lab protocol [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. After the generation of BMDMs treated with TMC in the absence/presence of doxycycline (20\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 24 hr. Naive total T-cells/CD4\u003csup\u003e+\u003c/sup\u003e T cells were added to the culture at a ratio of 1:10. Naive T cells without any stimulation served as a negative control. T cells were stimulated with CD3 and CD28 activating antibodies to analyse non-specific activation. After 3 days of co-culture in 96 well plates, IL-12, IFN-γ, IL-10 and TGF-β were quantified from the supernatant by ELISA. Simultaneously in a replicate experiment Brefildin-A was added to co-culture after 6hrs and incubated for 48hrs to analyse intracellular marker by flowcytometry or RT-PCR. The proliferation of T cells was measured either by MTT assays or CFSE proliferation assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Quantitative real time (RT)-PCR\u003c/h2\u003e \u003cp\u003eGene expression was carried out by stabilised lab protocol [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].Briefly, the total RNA was extracted using TRIzol method and quantified. 2\u0026micro;g of RNA was used for cDNA synthesis by iScript cDNA synthesis kit (Bio-Rad USA).β-Actin was amplified from each sample to ensure equal cDNA input.100 ng of cDNA was used for amplification of mentioned genes in triplicates using gene-specific primers \u003cb\u003e(supplementary table 3).\u003c/b\u003eUsing power SYBR green 2X Master Mix in applied Bio systems) Step one plus Thermal cycler for 40 cycles and analysed with SDS 2.4 software (Applied Bio systems).Results normalized according to the expression levels of GAPDH mRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western Blot:\u003c/h2\u003e \u003cp\u003eProtein expression was analysed by western blotting [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly Radio immuno precipitation Assay (RIPA) buffer Supplemented with Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor (PI) acceding to the manufacture\u0026rsquo;s recommended concentration was used to lysate cells. Each sample was normalized to 50 \u0026micro;g/30\u0026micro;l protein was resolved on an SDS-PAGE gel and electro-blotted onto a PVDF membrane. The membrane was blocked with 5% non-fat milk or 2% BSA in TBS for 2 h at RT. The membrane was probed overnight at 4˚c with following primary antibodies: sheep anti-mouse Arginase-1, Rabbit anti-mouse iNOS and GAPDH at (1:1000) dilutions. GAPDH was used as an internal control. Later, the membrane was probed with respective HRP- conjugated secondary antibody (1:5000) for 1 h at RT followed by the detection with ECL kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Flowcytometry analysis:\u003c/h2\u003e \u003cp\u003eCells were collected and stained for flow cytometry analysis. For In vitro experiment we used BMDMs for macrophage polarization experiment. BMDMs polarized in to M\u003csub\u003e0\u003c/sub\u003e, M\u003csub\u003e1\u003c/sub\u003eand M\u003csub\u003e2\u003c/sub\u003emacrophages [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]and treated with TMC for 24 h followed by doxycycline (20\u0026micro;g/ml) treatment for 24hrs and surface protein was analysed by flow cytometry. The cell were analysed for expression of: CD11b-PE, CD86-FITC and CD206-APC. Details dilution and brand are mentioned in (\u003cb\u003esupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor In vivo experiment we used Single cell suspensions from tumor infiltrating lymph nodes and tumor tissue, for intracellular staining, Brefeldin-A solution in DMSO had been added 12hrs before collection at a concentration of 1\u0026micro;g/ml. Positive control T cells were activated with CD28/3. Staining with CD44, CD39, CD73, CD11b, F4/80, CD86, CD206, CD3, CD4, CD8, FOXP3, Tbet, IFN-γ, IL-10, TGF-β (250x dilution each) was done using lab optimizing protocol [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Details dilution and brand are mentioned in (\u003cb\u003esupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/b\u003eCells were blocked with Fc block CD16/32 antibody for 30 min before staining as a background staining control. Everything was done on ice. The acquisition was done on Backman Coulter .Cytoflex research flow cytometer. Data were analyzed using CytExpert 2.4 from Beckman Coulter and FlowJo_V10. To quantify the cells recruited, equally weighed tissue was minced in the PBS and cells were collected in equal volumes of PBS. \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003e2.9 ELISA\u003c/span\u003e:\u003c/p\u003e \u003cp\u003eBMDM cells were seeded at a density of 10\u003csup\u003e5\u003c/sup\u003ecells/well in triplicates in 96-well plate and were treated was indicated. After24hrs cell free supernatants were analysed for cytokines by ELISA. 96-well plate were coated with the capture antibody in coating buffer (Carbonate buffer PH 9.1) overnight at 4˚C.The wells were washed with washing buffer (PBST with 0.05% Tween)for 5 times and block with blocking buffer (2.5% FBS in washing buffer).The collected cell free supernatants were added to the wells and incubated overnight at 4˚C.Later the wells were washed with buffer for 5 times and incubated with detection antibody (Biotin-labelled) in blocking buffer for 2hrs.After washing the wells were incubated with Streptavidin-HRP for 30 min in dark; following its incubation with TMB substrate for 15 min stop solution (2N orthophosphoric acid) was added to wells and reading were taken at 450nm.All the ELISA experiments were done in triplicates and O.D was used to deduced concentration of cytokines using standard graph[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Details dilution and brand are mentioned in (\u003cb\u003esupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 ATP Determination Assays:\u003c/h2\u003e \u003cp\u003e4T1 or BMDM cells were seeded into a 96-well plate at a density of 10,000 cells per well in 10% FBS containing media. The cells were treated with doxycycline 20\u0026micro;g/ml, in the presence of the Real Time-Glo\u0026trade; Extracellular ATP Assay Reagent (Promega, GA5010-1KT)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].Luminescence data was collected every 6 hours using a BMG POLAR star\u0026reg; plate reader\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Adenosine Assays:\u003c/h2\u003e \u003cp\u003eAdenosine was determined using culture supernatant of 4T1, BMDM and T cells samples by Adenosine Quantification Assay Kit (Sigma, MAK433-1KT) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].According to the manufacturing recommendations, we have used 20\u0026micro;L of frozen plasma samples to measure adenosine concentration. The plate was read on spectraMaxM2 Spectrofluorometer. The fluorescent product was excited at 535 nm and detected at 587 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2.12 Cytotoxic T-cell assay\u003c/span\u003e:\u003c/h2\u003e \u003cp\u003eFor CTL assay, in order to track proliferation of the 4T1 cells, 4T1 cells were stained with CFSE cell division tracker kit (Biolegend Cat#423801) according to the manufacturer\u0026rsquo;s protocol, Briefly,4T1 resuspended in PBS 10\u003csup\u003e6\u003c/sup\u003e cells per mL were stained with 2\u0026micro;l of 10mM CFSE per 10\u003csup\u003e6\u003c/sup\u003e cells to yield a final CFSE concentration of 1\u0026micro;M.4T1 cells were incubated at 37˚ C for 15 min, centrifuged at 500g for 8min,resuspended in RPMI to neutralize the Dye, and incubated at 37˚C for 30 min. Cells were centrifuged again, reseupended at 10\u003csup\u003e6\u003c/sup\u003e cells per mL, and stored at 37˚C until plating with the total T-cells. Later total T-cells were purified from splenocytes of different experimental groups using nylon wool column. Total T-cells were co-cultured with polarized macrophage as mentioned earlier.[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Later the primed T cells, harvested from each experimental group were plated with CFSE stained 4T1 at ratios of 1:100 for 5 days. These cells were centrifuged, the supernatants were stored at -20˚C for analysis of cytokines levels using ELISA and cells were stained with CD44-APC.The cytolytic activity of Tcells against 4T1 cells were analyzs by CFSE proliferation assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.13 4T1 Tumor model\u003c/h2\u003e \u003cp\u003eA syngenic orthotropic mouse breast cancer model was established using 4T1 cells as previously reported [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e][\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]Briefly, 4T1 cells (5*10\u003csup\u003e5\u003c/sup\u003ein PBS) were injected into BALB/C mice (n\u0026thinsp;=\u0026thinsp;5) through subcutaneously mammary gland. After tumor was palpable, the mice were treated with, Doxycycline (50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eof body weight), ARL67156 (2mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eof body weight) and AMPCP (20mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of body weight) was injected intraperitoneally in 50\u0026micro;L PBS on days 10, 13, 16, 19, 22, 25, 28, and 31th days. The tumor size was measured every alternate day using Vernier callipers, and the volume was calculated using the formula (length*width*width)/2. The mice were sacrificed on day 34 of tumor inoculation and excised tumors were weighed.\u003c/p\u003e \u003cp\u003e All experiments were conducted in accordance with the guidelines of the Institutional Animal Ethical Committee after obtaining a clearance at Nirma University, Ahmedabad India. Housing and handling of mice was in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) at Institute of Pharmacy Nirma University, Ahmedabad, India. Protocol No: \u003cb\u003eIP/PCOL/FAC/32/2022/49\u003c/b\u003edated 20/09/2022.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical tests:\u003c/h2\u003e \u003cp\u003eAll the data were collected and analysed from minimum of three individual biological experiment replicates including animal experiments. One-way ANOVA with Turkey\u0026rsquo;s test was used to analyse the significance between the groups. Two-way ANOVA with Turkey\u0026rsquo;s multiple comparison tests was used to analyse the difference among subgroups and the difference among groups; and longitudinal values. Significance was denoted as p. (*** means p\u0026thinsp;\u0026lt;\u0026thinsp;000.1; ** means p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; * means p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; n.s means not significant). In the figures * to denote the significance difference. These denotations were explained in the figure legend.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Doxycycline reverts TMC-induced M2 macrophages to M1 phenotype to induce Th-1 response\u003c/h2\u003e \u003cp\u003eIn our previous observations, we observed the anticancer potential of doxycycline on murine breast cancer cells (Supplementary Fig.\u0026nbsp;1A-I). Here, we wanted to check immunomodulatory potential of doxycycline on macrophage polarization. In order to generate TAMs in the \u003cem\u003ein vitro\u003c/em\u003e conditions, we have cultured bone-marrow derived macrophages (BMDMs) in tumor mimicking conditions (TMC) as explained in materials and methods (2.3).We have observed that TMC-induced CD11b\u003csup\u003e+\u003c/sup\u003e macrophages showed high expression of M2 markers- CD206, Arginase-1 and low expression of M1 markers- CD86 and iNOS indicating that TMC skewed macrophages to an immunosuppressive M2 phenotype. Later; we polarized the macrophages to M1 and M2 phenotype. Macrophage polarization was confirmed with increased CD206 expression in M2 macrophages and increased CD86 expression in M1 macrophages respectively (Sup. Figure\u0026nbsp;2A).Polarized macrophages were later incubated in TMC in the presence/absence of doxycycline. We have observed that doxycycline treatment significantly decreased the frequency of CD11b\u003csup\u003e+\u003c/sup\u003e CD206\u003csup\u003e+\u003c/sup\u003e M2 macrophages, while enhancingCD11b\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e M1 macrophages in TMC-induced immunosuppressive microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). It was further confirmed by iNOS and ARG-1 expression. We observed that increase in iNOS expression and subsequent decrease in IL-10 and TGFβ cytokines in TMC induced M2-polarized macrophages moreover decreased ARG-1 expression and subsequent decrease in IL-10 and TGFβ cytokines in TMC induced M1 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). Thus, doxycycline enhanced the frequency of anti-tumor M1 macrophages in tumor mimicking conditions. It is reported that distinguished macrophage subset differentiates CD4\u003csup\u003e+\u003c/sup\u003e T cells to a distinct T cell subset. In macrophage-CD4\u003csup\u003e+\u003c/sup\u003eT cell co-culture assay, we observed that doxycycline treatment enhanced CD4\u003csup\u003e+\u003c/sup\u003e T cell proliferation with subsequent increase in inflammatory T cell transcription factor Treg and cytokine (IFNγ) and decreased Foxp3 mRNA expression and immunosuppressive cytokines (IL-10, TGFβ) in both polarized macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-I).These results gave us an indication that doxycycline via enhancing M1 macrophages modulated the adaptive immune response in TME.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Doxycycline delayed tumor growth by decreasing the frequency of CD206\u003csup\u003e+\u003c/sup\u003e M2 macrophages in TME.\u003c/h2\u003e \u003cp\u003eThe above results highlighted the importance of doxycycline on macrophage polarization in TME. Further, we wanted to extrapolate these observations in \u003cem\u003ein vivo\u003c/em\u003e conditions using 4T1 induced carcinoma. We generated a syngeneic orthotropic breast cancer model using 4T1 breast cancer cells as explained in materials and methods (\u003cem\u003e2.13\u003c/em\u003e). Doxycycline treatment (50mg/kg of BW) was given for 8 doses starting from Day 10 of tumor injection, once the tumor was palpable. The mice were sacrificed on Day 34 and tumor tissues from control and doxycycline treatment group were excised (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).The weight and volume of tumor from both the groups were measured. The doxycycline group showed a significant decrease in tumor weight, volume and percentage of tumor regression compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). In order to understand the in vivo efficacy of doxycycline on macrophage polarization, we have checked the frequency of M1 (CD11b\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e) and M2 (CD11b\u003csup\u003e+\u003c/sup\u003e CD206\u003csup\u003e+\u003c/sup\u003e) macrophages in tumor tissue of control and treatment group. We have observed that tumor control had increased number of CD11b\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e M2 macrophages compared to CD11b\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e M1 macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). It was evident with increased release of IL-6, IL-10 and TGF-β cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). On the contrary, doxycycline treatment group showed decreased frequency of CD11b\u003csup\u003e+\u003c/sup\u003e CD206\u003csup\u003e+\u003c/sup\u003e macrophages in the tumor tissue, along with increase in TNFα, IFNγ, and IL-12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). These results were in accordance with the above obtained in vitro results. Later, we checked the frequency of CD4\u003csup\u003e+\u003c/sup\u003eand CD8\u003csup\u003e+\u003c/sup\u003e T cells from tumor infiltrating lymph nodes. Surprisingly, we observed that the tumor control had high number of CD4\u003csup\u003e+\u003c/sup\u003e T cells compared to CD8\u003csup\u003e+\u003c/sup\u003e T cells, while in doxycycline treatment group; the frequency of CD8\u003csup\u003e+\u003c/sup\u003e T cells is significantly increased from 10.3\u0026ndash;23.4%, quite crucial for promoting anti-tumor immunity. We also checked Cytokine level of lymph node supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ).Since, we observed the increased frequency of CD8\u003csup\u003e+\u003c/sup\u003eT cells in doxycycline treat group, we checked for anti-tumor cytotoxic potential by CTL assay. In CTL assay, the activated T cells isolated from tumor infiltrating lymph nodes were co-cultured with 4T1 breast cancer cells for 5 days and were checked for 4T1 proliferation. We observed an increased CTL activity of doxycycline group with decreased proliferation of 4T1 cells and increased release of IFNγ and TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK, L). Overall, this data suggested that doxycycline enhanced anti-tumor immunity by improving anti-tumor innate and adaptive immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Doxycycline promotes ATP release from cancer cells and alters downstream CD39 and CD73 expression on cancer and immune cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is reported that the balance between ATP release and ATP consumption is disturbed in TME, leading to the accumulation of extracellular ATP in the TME [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The extracellular ATP is hydrolysed by ectonucleotidase CD39 and CD73, metabolising it to adenosine which further aids in enhancing immune suppression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, inhibiting adenosine in TME aids in reduction of immune suppression in TME. Doxycycline is known to exhibit anti-tumor effect by targeting mitochondrial biogenesis and oxidative phosphorylation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. So, in order to elucidate the underlying mechanism of immune modulation by doxycycline in TME, we have focused on ATP metabolism in TME. We have observed that in breast cancer cells, there was an increase in ATP concentration upon doxycycline treatment on 18 hrs which gradually decreased in 36 hrs (Fig: 3A). Simultaneously, we observed that there was no significant difference in adenosine concentration in control and doxycycline treated breast cancer cells till 18 hrs. Interestingly, after 18hrs, an increase in adenosine concentration was observed in control cells but not in doxycycline treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).Similarly, the doxycycline treatment on TMC-induced macrophages also showed similar observations (Fig.\u0026nbsp;3Cand D), indicating that doxycycline manipulated ATP metabolism in TME to further understand its effect on ATP metabolism, we checked for downstream ectonucleotidase -CD39 and CD73 expression in a time-dependent manner. Interestingly, we observed that the expression of CD39 and CD73 was similar in untreated and doxycycline-treated cancer cells till 18 hrs. After 18 hrs, there was a decrease in the CD39 and CD73 expression in doxycycline treated cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F).This result aligned with our above observation of decreased adenosine concentration upon doxycycline treatment, indicating that doxycycline decreased CD73 and CD39 expression. Later, we extrapolated our findings to TMC-induced immunosuppressive macrophages. We observed that TMC-induced CD11b\u0026thinsp;+\u0026thinsp;macrophages had high expression of CD39 and CD73, which was decreased on doxycycline treatment in a time dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). These results indicate that doxycycline decreased CD39 and CD73 expression in a time dependent manner that in turn reduces adenosine levels to a lesser extent in TME. Additionally, we have checked the expression of ectonucleotidase in polarized macrophages. It was observed that immunosuppressive M2 macrophages showed high expression of CD73 and CD39 compared to anti-tumor M1 macrophages (SF, 4E). The frequency of CD39\u003csup\u003e+\u003c/sup\u003e and CD73\u003csup\u003e+\u003c/sup\u003emacrophages was increased in M1 and M2 macrophages when cultured in TMC. Doxycycline attenuated the frequency of CD39\u0026thinsp;+\u0026thinsp;CD73\u0026thinsp;+\u0026thinsp;TMC-induced macrophages in both polarized macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-L).Overall data suggested that TMC-induced macrophages depict an immunosuppressive M2 phenotype with high expression of CD39 and CD73, which are abrogated by doxycycline in TME.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antagonist of ectonucleotidase enhances doxycycline mediated anti-tumor immune response.\u003c/h2\u003e \u003cp\u003eEven though doxycycline restricts adenosine generation in TME, there is still a scope to the improve tumor weight and volume. In order to enhance the immunotherapeutic efficacy of doxycycline in context to ATP metabolism, we have tried to investigate the effect of doxycycline in combination with antagonist of CD39 (ARL67156) and CD73 (AMPCP). In murine breast cancer model, doxycycline (50mg/kg of BW), ARL67156 (2mg/kg of BW), AMPCP (20mg/kg of BW) were injected intraperitoneally in tumor bearing mice for 8 doses, every alternative day as explained in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). There was a significant reduction in the tumor weight and volume with no observed death in combination therapy of doxycycline with antagonist of ectonucleotidase (CD73 and CD39) in comparison to alone doxycycline/ CD39 and CD73 antagonists (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E).The inhibition of CD73 and CD39 by antagonists was confirmed with an increased in ATP concentration and decrease in adenosine levels in tumor tissue the reduction in adenosine levels was significantly high in the combination treatment of both antagonist along with doxycycline in comparison to doxycycline along with either antagonist (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and G). In TME, it is reported that cancer cells and various immune cells such as regulatory T cells, TAMs exhibit high expression of ectonucleotidase (CD39\u0026amp;CD73) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].So, we wanted to check the effect of antagonist and doxycycline treatment on ectonucleotidase in \u003cem\u003ein-vivo\u003c/em\u003e conditions. In CD44\u003csup\u003e+\u003c/sup\u003e breast cancer cells, we observed that the population of CD44\u003csup\u003e+\u003c/sup\u003e CD39\u003csup\u003e+\u003c/sup\u003e and CD44\u003csup\u003e+\u003c/sup\u003eCD73\u003csup\u003e+\u003c/sup\u003ecells was increased in tumor control, which on treatment with doxycycline and antagonist (ARL67156; AMPCP) was decreased to some extent. It was observed that combination of doxycycline along with antagonist limited CD73\u003csup\u003e+\u003c/sup\u003e CD39\u003csup\u003e+\u003c/sup\u003e population in the tumor tissue compared to alone treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u0026amp;I). Similar results were obtained in tumor infiltrating immune cells (macrophages and T cells). The infiltrating immune cells of tumor group had high CD39\u003csup\u003e+\u003c/sup\u003eCD73\u003csup\u003e+\u003c/sup\u003e expressing cells which were abrogated in combination treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-M).Interestingly; we have observed that CD39 expressing cells were high in infiltrating myeloid cells, while CD73 expressing cells were high in lymphoid cells of tumor control. All these results indicated a synergy between doxycycline and CD73 CD39 antagonists, resulting in decreased tumor progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Combination treatment of doxycycline and antagonist (ARL67156, AMPCP) potentiates anti-tumor macrophages subset in TME.\u003c/h2\u003e \u003cp\u003eAs in above observed results, the immunosuppressive M2 macrophages had an enhanced expression of CD73 and CD39 in comparison with M1 macrophages. So, we further wanted to check if this decrease in ectonucleotidase expression by combinational treatment skews tumor infiltrating macrophages to anti-tumor M1-phenotype. The tumor tissues from control and treatment groups were excised and stained for CD11b\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e M1 macrophages and CD11b\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e M2 macrophages. We observed that tumor group had significantly high frequency of M2 macrophages and low frequency of M1 macrophages. The tumor tissue of doxycycline treatment group had significantly enhanced frequency of M1 macrophages from 1.46\u0026ndash;11.8%as compared to ARL67156 and AMPCP alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The population of M1 macrophages were improved in combination treatments. The combination treatment of doxycycline along with both antagonists potentiated anti-tumor macrophages to almost 27.7% in the tumor tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We also observed increase the expression of iNOS from 5.05\u0026ndash;60.8% and decrease the arginase-1 from 76.3\u0026ndash;9.56% expression on combination treatment of doxycycline along with both antagonists. (Fig: 5B\u0026amp;C) The functional aspects of macrophages were confirmed by cytokine release. The excised tumor tissue from different groups were minced and checked for inflammatory and immunosuppressive cytokines. It was observed that supernatant of tumor control had high levels of IL-10 and TGF-β and low levels of IFN-γ, TNF-α. The levels of inflammatory cytokine (IFN-γ, TNF-α) were gradually increased from groups with alone treatment to combinational treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). This data were in coherence with the increase in M1 population in treatment groups indicating that the shift in cytokine milieu in TME is mediated by macrophage polarization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Antagonists of ectonucleotidase synergizes with doxycycline to attenuate regulatory T cells in TME.\u003c/h2\u003e \u003cp\u003eThe immunosurveillance by adaptive immune cells is essential to restrict tumor growth and development [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].Since we observed that doxycycline alone or in combination with both antagonists enhances innate immunity; we wanted to check its outcome on adaptive immunity. The total lymphocytes were isolated from tumor infiltrating lymph nodes of different experimental groups. The CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells were significantly increased in tumor control group than na\u0026iuml;ve from 26.7\u0026ndash;70.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Surprisingly, we observed that doxycycline along with antagonists of CD73 and CD39 in combinations had decreased the population of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells from 70.9\u0026ndash;26.6% as compare to along treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells subsets: T\u003csub\u003eH\u003c/sub\u003e-1, T\u003csub\u003eH\u003c/sub\u003e-2 and regulatory T cells in crucial in determining the outcome of tumor progression/regression [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recruitment of regulatory T cells in TME is one of major mechanism adopted by tumor cells for immune evasion [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e],[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Regulatory T cells are major contributors to extracellular adenosine-dependent immune suppression in TME [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].The lymph node of tumor control group showed low frequency of CD4\u0026thinsp;+\u0026thinsp;Tbet\u0026thinsp;+\u0026thinsp;IFN-γ\u003csup\u003elow\u003c/sup\u003e secreting T\u003csub\u003eH\u003c/sub\u003e-1subsets(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB \u0026amp;C);and high frequency of CD4\u0026thinsp;+\u0026thinsp;FOXP3\u0026thinsp;+\u0026thinsp;IL-10 \u003csup\u003ehigh\u003c/sup\u003e + TGFβ \u003csup\u003ehigh\u003c/sup\u003e regulatory T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F).In treatment groups, enhanced T\u003csub\u003eH\u003c/sub\u003e-1 population as evident with increase in CD4\u003csup\u003e+\u003c/sup\u003eTbet\u003csup\u003e+\u003c/sup\u003e IFNγ \u003csup\u003ehigh\u003c/sup\u003e T cell subsets which were further improved in combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026amp;C). On the other hand, we have observed that treatments showed decrease in frequency of CD4\u003csup\u003e+\u003c/sup\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e TGFβ\u003csup\u003ehigh\u003c/sup\u003e regulatory T cells which was improved in combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F).It was further confirmed with decrease in released IL-10 and TGFβ cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026amp;J).These data were additionally confirmed with increase in IFNγ and TNFα analysed from the supernatants of tumor draining lymph node (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026amp;H). All these data indicate tumor group consist of high frequency of regulatory T cells while doxycycline and antagonists alone/in combination skew the ratio of pro-tumor immunity to anti-tumor immunity by enhancing Th1 phenotype. The combination treatment of both antagonists along with doxycycline exerts a better immune outcome than alone treatments/ in combination with either antagonist.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Combination treatment exerts an immune protective response by enhancing CD8 T cells mediated cytotoxicity\u003c/h2\u003e \u003cp\u003eThe studies have reported a positive correlation between infiltrating CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells and enhanced survival of cancer patients [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e],[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we have observed that doxycycline enhanced the frequency of CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells rather than CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells in tumor bearing mice. As we observed a decrease in CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cell population in treatment groups of tumor bearing mice, we checked the population of CD8\u003csup\u003e+\u003c/sup\u003e T cells in treatment groups of tumor bearing mice. We observed that not only doxycycline but CD73 and CD39 antagonists also enhanced CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells population with subsequent IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). The immunosuppressive factors in TME are reported to mediate CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cell exhaustion with low cytotoxic activity. It is reported thatCD39 is a signature marker for CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cell exhaustion [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].Interestingly, inCD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells, we have observed a reduction in CD39 and CD73 expression in doxycycline alone or in combination with antagonists of tumor bearing mice compared to the tumor control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026amp;D).The ultimate immune outcome in cancer regression is mediated by cytolytic activity of activated CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells. In order to confirm the functional aspect of CD8\u003csup\u003e+\u003c/sup\u003e T cells. We checked for anti-tumor cytotoxic potential by CTL assay. In CTL assay, total T cells were isolated from lymph node of different experimental groups and co-cultured with 4T1 breast cancer cells. We have observed that doxycycline along with antagonist of CD39 and CD73 combinational group showed decreased viability of 4T1 cells that is from 89.5\u0026ndash;32.0% moreover its increased cytokines level of IFN-γ and TNFα and decreased cytokine level of IL-10 and TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-I) Overall results indicated that doxycycline or antagonists of CD73 and CD39 alone skewed the immune response to antitumor immunity but when given in combination, mediated a better synergy and exerts an efficient innate and adaptive anti-tumor immune response, making it a promising immunotherapeutic strategy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we have demonstrated the immune modulation potential of doxycycline in TME and the importance of doxycycline repositioning with metabolic checkpoint inhibition in TME. Our study highlights the usage of combinational therapy of doxycycline and antagonists of CD39 and CD73 to improve clinical outcomes in triple negative breast cancer (TNBC). Triple negative breast cancer is highly prevailing disease worldwide with no promising treatment modality [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e][\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e][\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e][\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Hence, identification of novel therapeutic approach that could potentially impact the disease is quite essential. Monocytes in tissue microenvironment differentiate to either inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Inflammatory macrophages are immune-stimulatory, important for clearance of infection, while M2 subset aids in tissue repair and maintaining homeostasis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In TME, macrophages, on encounter with tumor-derived factors, polarize to M2-like subset termed as TAMs. An abundant infiltration of TAMs is highly correlated with the severity of the disease progression [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e][\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. TAMs play a pivotal role in mediating immune evasion and are responsible for the failure of immunotherapeutic treatments. Thus, strategies targeting repolarization of TAMs to anti-tumor subset have received a great attention [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].Tetracyclines like doxycycline have been explored for their anti-cancer potential in several cancer models [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e][\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e][\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is reported to decrease the tumor growth and delay the reoccurrence of tumor in murine models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Recently, the studies are focused on understanding the immune modulatory potential of doxycycline. In the model of choroidal neovascularization, doxycycline inhibits M2-polarization in dose-dependent manner [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e][\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we have investigated the immune modulatory potential of doxycycline on macrophage polarization in TME. We have observed that doxycycline treatment inhibited CD206\u0026thinsp;+\u0026thinsp;TAMs in tumor tissue of TNBC. Additionally, we have observed that doxycycline treated mice showed reduced tumor growth and enhanced CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell frequency compared to control mice. This finding suggests that doxycycline potentiated anti-cancer efficacy by enhancing host anti-tumor immune responses.\u003c/p\u003e \u003cp\u003eOne of the study has reported the important use of doxycycline in combination with chemotherapeutic drugs is because of its ability to target mitochondria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The treatment with doxycycline induces mitochondrial dysregulation by altering membrane potential that ultimately leads to apoptotic cell death [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Upon apoptotic cell death, ATP is released in the extracellular microenvironment, affecting cellular metabolism in TME [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e][\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We have observed that treatment with doxycycline on TNBC promoted the release of ATP compared to control cells. In TME, the extracellular ATP is degraded to adenosine by ectonucleotidase (CD39 and CD73) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The over expression of ectonucleotidase has been well documented in various cancer models. In TNBC patients, the ectonucleotidase CD39 and CD73 are highly expressed on tumor cells and infiltrating immune cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] The generated adenosine further contributes to immune suppression by inhibiting antigen presentation, generation of TAMs and recruitment of regulatory T cells[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e][\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Numerous studies have shown that CD39-/- or CD73-/- mice inhibited tumor growth and experimental metastasis, suggesting that ATP/CD39-CD73/adenosine axis is important for immune suppression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, we have observed that doxycycline treatment decreased adenosine levels on 4T1 cells and 4T1-induced immunosuppressive macrophages after 18 hrs of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and D). So, we have explored the time-dependent expression of ectonucleotidase and effect of doxycycline on intrinsic CD39 and CD73 expression in tumor and infiltrating immune cells. We have observed that intrinsic expression of CD39 and CD73 in 4T1 cells increased after 18 hrs (SF.4 A-C). Additionally, we have observed doxycycline treatment partially abrogated the CD39 and CD73 expression on 4T1 and M2 macrophages (which had high expression compared to M1 macrophages). Thus, our data revealed that doxycycline exhibited immunotherapeutic efficacy by suppressing ectoenzyme mediated generation of TAMs and subsequent immune suppression in TME.\u003c/p\u003e \u003cp\u003eIn various cancer cell model, preclinical and clinical trials of small molecule inhibitors of ectonucleotidase CD39 and CD73 promoted anti-tumor immunity by stimulating APCs and restoring IFNγ secreting T cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Although doxycycline partially decreased the expression of CD39 and CD73, but absolute decrease in their expression would attenuate tumor growth and improve the overall survival. So, in this study, we tried to explore the combination effect of doxycycline along with the antagonists of CD73 (AMPCP) and CD39 (ARL67156). The combinational therapy of doxycycline and antagonists significantly decreased the tumor growth and enhanced the overall survival of tumor bearing mice. It also reduced the expression of CD39 and CD73 on tumor and infiltrating immune cells (macrophages and T cells), resulting in decreased adenosine levels in TME. As in observed results, the immunosuppressive M2 macrophages had an enhanced expression of CD73 and CD39 in comparison with M1 macrophages [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. So, we further wanted to check if this decrease in ectoenzyme expression by combinational treatment skews tumor infiltrating macrophages to anti-tumor M1-phenotype Subsequently, we have observed an increase in the frequency of F4/80\u0026thinsp;+\u0026thinsp;CD68\u0026thinsp;+\u0026thinsp;M1 in the combinational therapy of doxycycline and antagonists compared to individual components.\u003c/p\u003e \u003cp\u003eIn TME, regulatory T cells consume ATP to produce adenosine that via A2AR/A2B receptor activation inhibits effector CD4 and CD8 T cells, generating T cell anergy [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e][\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].In addition to this, the generated adenosine aids in expansion of regulatory T cells by A2AR activation on regulatory T cells [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e][\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]Thus, adenosine maintains a positive feedback loop to establish immunosuppressive microenvironment [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The doxycycline alone or in combination with antagonist of ectonucleotidase therapy promotes the ratio of Foxp-3\u003csup\u003e+\u003c/sup\u003e IL-10\u003csup\u003e+\u003c/sup\u003e regulatory T cells/Tbet\u003csup\u003e+\u003c/sup\u003e IFN-γ\u003csup\u003e+\u003c/sup\u003e Th1 to Th1 phenotype shifting balance of T cells from immune evasion to immunosurveillance. Thus, our study provides strong evidence that repositioning doxycycline alone or in combination with antagonist of ectonucleotidase. Significantly reduced the tumor growth and promoted immune surveillance in TME. Thus, we report that doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthor contribution:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePD designed and performed the experiments; analysed the data; prepared figures; drafted the manuscript and revisions. DS helped in animal experiments and analysed data. KS, KT helped in animal experiments. JS \u0026amp; CD extended animal facility, RAR conceived the study, designed the experiments and prepared and finalised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgement:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank Dr. Abhijit de, ACTREC, Mumbai for providing TNBC-4T1 cell line. PD fellowship is supported by Indian Council of Medical Research (ICMR) and ScHemeOf Developing High quality research (SHODH); KS and SY fellowship were supported by Indian Council of Medical Research (ICMR); DS fellowship is supported by Lady Tata Memorial Trust (LTMT). This study was financially supported by Gujarat State Biotechnology Mission (GSBTM). Authors acknowledge Mansi Vaghela Hima Vora, Miloni Mehta and for their support in routine lab work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConflict of Interest:\u003c/u\u003e\u003c/strong\u003e There is no conflict of interest among the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe have declared that all experiments were conducted according to the guidelines of the Institutional Animal Ethical Committee after obtaining a clearance at Nirma University, Ahmedabad India. Protocol No: \u003cstrong\u003eIP/PCOL/FAC/32/2022/49\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have declared that this study was financially supported by the Gujarat State Biotechnology Mission (GSBTM), Department of Science \u0026amp; Technology and Government of Gujarat with Grant No. \u003cstrong\u003eGSBTM/JD (R\u0026amp;D)/610/20-21/345.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWhiteside, T. The tumor microenvironment and its role in promoting tumor growth. \u003cem\u003eOncogene \u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 5904\u0026ndash;5912 (2008). https://doi.org/10.1038/onc.2008.271\u003c/li\u003e\n\u003cli\u003eHinshaw, D. C., \u0026amp;Shevde, L. A. (2019). 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The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center. \u003cem\u003eThe Journal of biological chemistry\u003c/em\u003e, \u003cem\u003e292\u003c/em\u003e(4), 1211\u0026ndash;1217. https://doi.org/10.1074/jbc.C116.764043\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Doxycycline, ectonucleotidase, tumor microenvironment, macrophage polarization, T cells, CD39, CD73, ARL67156 and AMPCP","lastPublishedDoi":"10.21203/rs.3.rs-3977928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3977928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eStudies have reported that cellular metabolism at tumor-immune microenvironment (TiME) serve as a critical checkpoint and perturbs/supports anti-cancer immunity. Extracellular ATP (eATP) may mediate anti-cancer immune response however; its catabolism by ectonucleotidase generates immunosuppressive adenosine. Antagonist of ectonucleotidases: CD39 and CD73 have been explored as potential therapeutic. In the presented work we have tried to repurpose doxycycline for mitigating ATP metabolism with or without antagonist of ectonucleotidase.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eeATP and adenosine level were quantified. The bone marrow-derived M1 and M2 polarized macrophages were maintained in tumor mimicking condition (TMC). Total or CD4\u003csup\u003e+\u003c/sup\u003e Tcells were co-cultured with macrophages to understand the impact of doxycycline and antagonist of ectonucleotidase T cell/subset differentiation. Preclinical efficacy of doxycycline and ectonucleotidase antagonist and their synergy was scored in 4T1 breast carcinoma.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDoxycycline manipulates macrophage polarization by decreasing the frequency CD206\u003csup\u003e+\u003c/sup\u003eM2 macrophages that promoted CD4\u003csup\u003e+\u003c/sup\u003e directed CD8\u003csup\u003e+\u003c/sup\u003e T cell mediated tumor cell lysis. Doxycycline alleviated the expression of CD39 and CD73, rescuing ATP catabolism. Doxycycline delayed tumor growth by enhancing F4/80\u003csup\u003e+\u003c/sup\u003e CD86\u003csup\u003e+\u003c/sup\u003e M1 macrophages and subsequently anti-tumor Tbet\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T-cells, attenuating the frequency of FOXP3\u003csup\u003e+\u003c/sup\u003e regulatory T cells which was cooperatively supported by ARL67156 and AMPCP (CD39 and CD73 antagonist). Doxycycline promoted CD8\u003csup\u003e+\u003c/sup\u003eT cell mediated cytotoxicity which was synergistically enhanced with ARL67156 and AMPCP ensuring a possibility of using doxycycline alone or in combination with antagonist of ectonucleotidase.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePresented data indicate a prospective usage of doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB.\u003c/p\u003e","manuscriptTitle":"Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 04:03:03","doi":"10.21203/rs.3.rs-3977928/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":"1c53a70d-e25b-42eb-8127-e8e98619d34c","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-01T04:59:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 04:03:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3977928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3977928","identity":"rs-3977928","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}