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Abstract
Background Immunotherapy, including adoptive cell therapy (ACT) and immune checkpoint inhibitors (ICIs), has a limited effect in most patients with colorectal cancer (CRC), and the efficacy is further limited in patients with liver metastasis. Lack of antitumor lymphocyte infiltration could be a major cause, and there remains an urgent need for more potent and safer therapies for CRC.
Methods In this study, the antitumoral synergism of low molecular weight heparin (LMWH) combined with immunotherapy in the microsatellite stable (MSS) highly aggressive murine model of CRC was fully evaluated.
Results Dual LMWH and ACT objectively mediated the stagnation of tumor growth and inhibition of liver metastasis, neither LMWH nor ACT alone had any antitumoral activity on them. The combination of LMWH and ACT obviously increased the infiltration of intratumor CD8+ T cells, as revealed by multiplex immunohistochemistry, purified CD8+ T-cell transfer assay, and IVIM in vivo imaging. Mechanistically, evaluation of changes in the tumor microenvironment revealed that LMWH improved tumor vascular normalization and facilitated the trafficking of activated CD8+ T cells into tumors. Similarly, LMWH combined with anti-programmed cell death protein 1 (PD-1) therapy provided superior antitumor activity as compared with the single PD-1 blockade in murine CT26 tumor models.
Conclusions LMWH could enhance ACT and ICIs-based immunotherapy by increasing lymphocyte infiltration into tumors, especially cytotoxic CD8+ T cells. These results indicate that combining LMWH with an immunotherapy strategy presents a promising and safe approach for CRC treatment, especially in MSS tumors.
- gastrointestinal neoplasms
- lymphocytes, tumor-infiltrating
- immunotherapy, adoptive
- immune checkpoint inhibitors
- tumor microenvironment
Data availability statement
Data are available upon reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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- gastrointestinal neoplasms
- lymphocytes, tumor-infiltrating
- immunotherapy, adoptive
- immune checkpoint inhibitors
- tumor microenvironment
WHAT IS ALREADY KNOWN ON THIS TOPIC
As an anticoagulant, low molecular weight heparin (LMWH) exhibits antiangiogenic activity by targeting vascular endothelial growth factor and other proangiogenic mediators, thereby suppressing both primary tumor growth and metastasis. Prior studies have demonstrated that antiangiogenic agents can normalize the tumor vasculature and reverse the immunosuppressive tumor microenvironment, thereby enhancing the efficacy of immunotherapy, however, patients with microsatellite stable (MSS) colorectal cancer (CRC) with liver metastases benefited minimally from the combination of antiangiogenesis plus immunotherapy. Given this, further exploring the therapeutic potential of LMWH combined with immunotherapy is warranted.
WHAT THIS STUDY ADDS
Using mouse models exhibiting excluded and inflamed tumors, we assessed the antitumor effects of immunotherapy alone or combined with LMWH. Our preclinical data showed that LMWH synergized with adoptive cell transfer or anti-programmed cell death protein 1 (PD-1) therapy to produce an enhanced antitumor activity against MSS CRC, even in liver metastatic models, which was attributed to tumor vascular normalization and increased antitumor T-cell infiltration. Given the observed synergism, further clinical evaluation of this LMWH combined strategy is warranted.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
These findings demonstrate that LMWH synergizes with immunotherapy to provide a potentially effective and safe therapeutic strategy for CRC, particularly MSS tumors. This combined approach may profoundly impact clinical management.
Introduction
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide,1 and around 40–50% of patients with CRC die from distant metastases.2 Current treatment for CRC consists mainly of surgery and chemotherapy, combined with targeted therapies such as monoclonal antibodies against vascular endothelial growth factor (VEGF) and epidermal growth factor receptor, if necessary.3 However, the 5-year survival rate remains low due to the acquired drug resistance in adjuvant therapies followed by unresectable metastases and tumor recurrence. Thus, novel and effective treatment modalities are emphasized to improve the prognosis of patients with CRC in the advanced stage.
Immunotherapy, including adoptive cell transfer therapy (ACT) and immune checkpoint inhibition, has mediated a great objective response in hematological tumors and some solid tumors and provides new ideas for advanced CRC treatment.4 Unfortunately, most patients with CRC respond poorly to immunotherapy. For ACT-based immunotherapy, many preclinical and clinical studies have reported poor penetration of exogenously infused antitumor T cells and limited immunogenic response in tumors.5–9 This inefficient delivery of T cells is probably due to the high interstitial fluid pressure (IFP),10 the pathological vascular network,11 12 the “physical barriers” filled with fibroblasts and extracellular matrix (ECM) in tumors,8 as well as the immunosuppressive tumor microenvironment (TME).13 For immune checkpoint inhibitors (ICIs), good clinical remission occurs only in a minority (15%) of patients with mismatch repair-deficient or microsatellite instability-high (dMMR/MSI-H). In contrast, the majority of patients with mismatch repair-proficient or microsatellite stability (pMMR/MSS) are almost unresponsive to ICIs.14 In the pMMR/MSS population, low tumor mutation burden (TMB) and immunogenicity result in the lack of immune cell infiltration, posited as mechanisms of immune resistance.15–17 Therefore, how to improve immune cell infiltration and expand the application of immunotherapy to patients with low and unresponsive CRC needs to be solved.
Low molecular weight heparin (LMWH) has been reported to inhibit tumor progression by coagulation-dependent as well as coagulation-independent mechanisms.18 By interfering with the coagulation cascade, LMWH reduces the formation of thrombin and fibrin, thereby preventing tumor growth and metastasis. Moreover, antiangiogenesis targeting VEGF and other proangiogenic factors, mediated by LMWH-released tissue factor pathway inhibitor, and the block of P- or L-selectin-mediated interactions between platelets and tumor cells by LMWH reduce both local tumor growth and metastasis.18–21 Two clinical trials in small cell lung cancer demonstrated significantly better survival in groups receiving chemotherapy plus LMWH than those treated with only chemotherapy.22 However, no studies have evaluated the therapeutic effect of LMWH combined with ACT or ICIs-based immunotherapy in solid tumors. Although current antiangiogenic agents can induce the normalization of tumor vasculature and reverse the immunosuppressive TME, thereby increasing immune cell infiltration and enhancing the efficacy of immunotherapy,23–27 the side effects, such as hypertension, proteinuria, skin and liver damage, and the risk of treatment-related thrombosis, are greatly increased when used together.28–30 Moreover, recent clinical data reported that patients with pMMR/MSS CRC with liver metastases benefited minimally from the combination of antiangiogenesis plus programmed cell death protein 1 (PD-1) blockade, whose response rate and overall survival were much lower than those without liver metastases or with only lung metastases, suggesting that the combination strategy is not as effective in patients with liver metastases.31 32 Based on this context, our study investigates whether LMWH in combination with ACT or ICIs could be a more effective and safer tumor conditioning strategy that may promote the introtumor lymphocytes infiltration and enhance the efficacy of cancer immunotherapy, thereby expanding the use of immunotherapy in CRC to a wider range of metastatic pMMR/MSS populations.
Methods
Cell lines and mice
The murine syngeneic MC38 colon adenocarcinoma cell line (Cat#ZQ0933, provided by Zhong Qiao Xin Zhou Biotechnology) and CT26 undifferentiated colon carcinoma cell line (Cat# CL-0071, provided by Procell Life Science & Technology) were used. Both cell lines were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) with 10% fetal bovine serum (Biological Industries), 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco) and maintained in an incubator with a humidified atmosphere of 5% CO2 at 37°C. All cell lines regularly tested negative for Mycoplasma contamination.
C57BL/6J and Balb/c mice were purchased from Hunan SJA Laboratory Animal. All experimental mice were bred and housed on a 12 hours light/dark cycle at a temperature of 22°C with 55% humidity and under specific pathogen-free conditions, in accordance with the animal experimental guidelines of the South China University of Technology. All animal procedures were approved by the Institutional Animal Care and Use Committee of the South China University of Technology.
In vitro lymphocyte activation and adoptive cell transfer
The day before ACT, lymphocytes were harvested from the spleens of C57BL/6J or Balb/c mice. Mouse spleens were gently ground using slides in phosphate-buffered saline (PBS) and passed through a nylon filter (200 mesh), and lymphocytes were separated using centrifugation at 450 g×5 min at room temperature. Red blood cells were depleted by red blood cell lysis (Beyotime, Cat# C7302). Lymphocytes were further cultured and activated in complete RPMI-1640 medium in the presence of 10 µg/mL concanavalin A (conA) (Sigma, Cat# C2010) and 500 IU/mL murine interleukin (IL)-2 (PeproTech, Cat# 212-12-5) for 24 hours. On the day of ACT, in vitro-activated lymphocytes (2×107) were transferred into tumor-bearing mice via the caudal vein. For ACT treatment with CD8+ T cells, 2×106 CD8+ T cells or non-CD8+ T cells sorted by murine CD8α (Ly-2) MicroBeads (Miltenyi, Cat# 130-117-044) after MACS were transferred. ACT treatment was performed within 24 hours after each LMWH administration. In experiments using subcutaneous tumor models, ACT was conducted on days 8, 11, 14 and 17 after tumor cell injection. Alternatively, in liver metastasis models, ACT was conducted on days 11, 14, 17 and 20 after liver metastases developed.
Tumor models and in vivo treatment
For the subcutaneous colorectal tumor model, MC38 and CT26 tumors were generated by injecting 5×105 and 1×106 cancer cells into the right flanks of C57BL/6J and Balb/c mice (6–8 weeks of age). Cells were suspended in 150 µL PBS with 50% Matrigel. LMWH administration was performed on days 4, 7, 10, 13, and 16, followed by ACT as described previously. The tumor volume (L×W2/2) and weight were measured every three days from day 7. Mice were sacrificed on day 19, and subcutaneous tumors were harvested. For the liver metastasis model, mice were anesthetized with 1.25% tribromoethanol (Nanjing Aibei Biotechnology, Cat#M2920) at 200 µL/10 g body weight before surgery, and then 6×105 CT26 cells suspended in 250 µL PBS were injected into the superior mesenteric vein via laparotomy. Mice that underwent surgery were allowed to recover for 4 days (day 4) before being treated intraperitoneally (i.p.) with LMWH every 3 days, and ACT was performed as described previously for metastases models. Mice were sacrificed on day 22, and body weights and liver weights were recorded. Representative liver tissue (right anterior, right middle, or left anterior) from each experimental group was collected, fixed with 10% formalin, dewatered, and embedded in paraffin.
LMWH (provided by National Institutes for Food and Drug Control, Beijing, China, Cat# 9041-08-1) was administered at a dose of 10 µg/g body weight. In the experiment in which ACT was combined with anti-PD-1 treatment, mice were i.p. treated with the in vivo blocking antibody of mouse PD-1 (Clone: 29F.1A2, Bio X Cell, Cat# BP0273) or the rat IgG2a antibody, which was the isotype control (Clone: 2A3, Bio X Cell, Cat# BP0089). Each PD-1 blockade was performed within 24 hours after LMWH administration, and the therapeutic doses were both 150 µg/per mouse (on days 8, 11, 14, and 17 post subcutaneous tumor challenge; on days 11, 14, 17, and 20 post liver metastasis challenge).
Detection of hypoxia
To visualize hypoxia, mice bearing CT26 tumors treated with or without LMWH were injected i.p. with 60 mg/kg pimonidazole (HP6-100kit; Hypoxyprobe) 1.5 hours before being sacrificed, and hypoxia was imaged in tumor sections by immunofluorescence (IF) staining with fluorescein isothiocyanate (FITC) -conjugated anti-pimonidazole antibody (Hypoxyprobe).
TUNEL staining
After MC38 or CT26 tumors were treated with LMWH combination treatment, paraffin sections were prepared by conventional methods and then underwent TUNEL staining using the TUNEL Apoptosis Detection Kit (KeyGEN BioTECH) according to the manufacturer’s instructions.
Multiplex immunohistochemistry staining
Multiplex immunohistochemistry (mIHC) was performed using the PANO Multiplex IHC kit (Panovue, 10144100100). Formalin-fixed paraffin-embedded sections (3.5 µm) were deparaffinized and rehydrated. Each slide underwent several cycles of staining, including heat-induced epitope retrieval with citrate buffer (pH=6.0) or Tris/EDTA (pH=9.0), endogenous peroxidase blocking with 3% H2O2, and non-specific protein blocking with 10% goat serum, followed by incubation of primary antibodies and corresponding horseradish peroxidase-conjugated secondary antibody (Panovue, 10013001040). Finally, tyramide signal amplification dyes were applied to amplify fluorescence signals. The following primary antibodies were used in sequential rounds of staining: HIF-1α (SANTA CRUZ, Cat#sc-13515, 1:200), CD8α (CST, Cat#98941, 1:500), PD-1 (CST, Cat#84651, 1:400), CD31 (CST, Cat#77699, 1:500), α-SMA (ProteinTech, Cat#14 395–1-AP, 1:12000), Ki67 (Abcam, Cat#ab16667,1:200), GZMB (CST, Cat#44153, 1:200), CXCR3 (Abcam, Cat#ab288437, 1:500), CXCL10 (ProteinTech, Cat#10 937–1-AP, 1:500). Slides were then counterstained with DAPI (Beyotime, C1005) and mounted with antifade mountant (Panovue, 10022001010).
Imaging and HALO analysis
For IF and mIHC, whole slides were scanned and digitalized by the Vectra Polaris Automated Pathology Imaging system (Akoya Biosciences, V.1.0). For H&E staining, images were acquired by the Aperio CS2 Digital Pathology scanner (Leica). Each slide was scanned at 20× objective magnification. A total of two to three fields with the same size were selected randomly on each slide of the tumor sample for analysis. Multispectral images were analyzed by HALO software (Indica Labs, V.3.3.25) with HighPlex-FL settings, IHC images were analyzed by mIHC settings, and H&E images were classified and quantified by classifier settings.
Cytotoxicity assays
MC38 or CT26 cells were cultured in 96-well plates (4×103 cells/per well) with or without LMWH (0.125, 0.25, 0.5, 1, 2 and 4 µg/µL), cocultured or uncocultured with lymphocytes (activated/non-activated) at effector:target ratios of 1:1 and 5:1 at 37°C for 12 hours. Next, the medium and lymphocytes were removed, and fresh medium was added to test wells. A CCK8 kit (Dojindo, Cat#CK04) was used to determine the cytotoxicity of LMWH or lymphocytes to tumor cells. Tumor cell viability was calculated by the following formula: viability=((a value in experimental well-a value in blank well)/(a value in control well-a value in blank well))×100%. Lymphocyte cytotoxicity was calculated by the following formula: cytotoxicity=((a value in control well-a value in experimental well)/(a value in control well-a value in blank well))×100%. The value was tested at OD/450 nm with a high-quality microplate reader (BioTek, 800TS).
Flow cytometry
To determine whether LMWH induces CD69 and PD-1 expression on T cells, murine spleen lymphocytes from Balb/c mice were stained with the following antibodies: anti-CD45 BV510 (BioLegend, Cat# 103137), anti-CD3 APC (BioLegend, Cat# 100236), anti-NK1.1 PE-Cy7 (BioLegend, Cat# 108922), anti-CD4 PerCP-Cy5.5 (BioLegend, Cat# 100434), anti-CD8 FITC (BioLegend, Cat# 126606), anti-CD69 BV421 (BD, Cat# 562920), and anti-PD-1 PE (BioLegend, Cat# 135206) antibodies. Nuclei were stained with DAPI (Beyotime, Cat# C1005). Cells were resuspended and blocked with CD16/32 (BioLegend, Cat# 101302) diluted in 1×PBS with 0.2% bovine serum albumin. Cells were detected by multicolor flow cytometry (BD, LSRFortessa) and analyzed by FlowJo (V.10.5.3).
MACS CD8+ T cells
Primary lymphocytes were isolated from Balb/c mouse spleens and activated by conA and IL-2 in vitro as previously described. Murine CD8α (Ly-2) MicroBeads (Miltenyi, Cat# 130-117-044), a MiniMACS separator (Miltenyi, Cat# 130-042-102), MS separation columns (Miltenyi, Cat# 130-402-201) and MACS MultiStand (Miltenyi, Cat# 130-402-303) were used for CD8+ T-cell purification. MACS was performed according to the Miltenyi MACS protocol; 95 µL MACS buffer was used for resuspension, and 10 µL microbeads were used for magnetic labeling every 1×107 cells at 4°C for 15 min. CD8+ T cells and non-CD8+ T cells were then separated after MACS and counted for transfer. Flow cytometric analysis was used to confirm that the CD8+ T-cell population purity was over 90% (BD, LSRFortessa).
Intravital two-photon microscopy
To confirm the distinctive effects of LMWH administration on circulating CD8+ T-cell migration and infiltration into tumors, the intravital two-photon microscopy platform (IVM-MS model, IVIM Technology) was used to visualize and examine this procedure. Balb/c mice bearing CT26 grafts received i.p. LMWH two times on day 4 and day 7. ACT using carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD8+ T cells was then performed on day 7. After 5 hours of ACT, blood vessel dye containing 10 mg/mL Evans blue (Absin, Cat#abs47002009) was administered (8 mL/kg body weight) via the caudal vein for vascular imaging. An hour later (6 hours), after blood vessel dye fully diffused into the circulation, mice were anesthetized, followed by tumor exposure, and imaged by two-photon microscopy. The same procedures were performed, and representative images and videos were captured at 12 hours, 24 hours and 48 hours after ACT. Three or four mice per group were used at every capture point, yielding similar results. Water objective lens (20×), green fluorescent protein (GFP) (498–542 nm, for CFSE) and near-infrared (NIR) (619–676 nm, for Evans blue) channels were used for the imaging model at 50% excitation of laser power.
Statistical analysis
All statistical data are presented as the mean±SEM. All sample sizes were large enough to ensure proper statistical analyses, which were performed using IBM SPSS V.25.0 and GraphPad Prism V.9.0. Two-tailed unpaired Student’s t-test was used when conditions of normality and variance homogeneity were met; otherwise, the Mann-Whitney test was used. Results with a p value<0.05 was considered statistically significant (*p<0.05; **p<0.01).
Results
LMWH treatment enhanced the antitumor efficacy of lymphocytes transfer therapy in tumor growth and liver metastasis
Tumor-infiltrating lymphocytes (TIL) localizing into the tumor is an absolute requirement for the success of ACT.33 In terms of density and localization of TIL, CRC can be categorized as an inflamed or excluded phenotype.34 “Inflamed” represents the abundance of TIL, especially CD8+ T cells, infiltrated in tumors that have high TMB and a pre-existing antitumor response. Whereas tumors with high TIL density in the invasive edge and low TIL infiltration in the core are defined as “excluded”, which have a limited immune response and are the most common immunophenotype in CRC. In our study, syngeneic murine CT26 MSS and MC38 MSI colon cancer models were used to represent these two distinct immunotypes. From IHC staining, CT26 tumors were characterized by CD8+ T cells restricted to the periphery and stroma and were similar to excluded-type, while MC38 tumors showed prominent CD8+ T cells in both the edge and core, which were close to inflamed-type (online supplemental figure S1A,B).
Supplemental material
Next, using subcutaneous CT26 and MC38 cancer models, we assessed whether the antitumor efficacy of ACT could be enhanced by LMWH treatment (online supplemental figure S1C). Results showed that only LMWH treatment combined with ACT significantly inhibited tumor growth in both CT26 and MC38 tumors, while neither LMWH nor ACT alone had an obvious therapeutic effect (figure 1A–F). Consistently, in the CT26 liver metastasis model, LMWH in combination with ACT produced a synergistic inhibition of liver metastases (figure 1G,H). Liver weight, the ratio of liver weight to body weight, and the ratio of metastasis area to total liver area were all significantly reduced in the combination group, while LMWH or ACT alone did not demonstrate any inhibitory effect (figure 1I–K). Similar results observed in both ectopic and metastatic models revealed that only combination therapy could exert significant antitumor activity, as compared with monotherapies. For ACT alone, the ineffective delivery of lymphocytes caused by barriers (eg, substantial IFP, high tissue stiffness, and dysfunctional blood vessels) in solid tumors could be an important reason for the inefficacy of adaptive therapy. For LMWH treatment alone, the antimetastatic property as previously reported was not observed in our study,35 which was probably related to the dose, frequency, and duration of LMWH administration.
LMWH treatment enhanced the antitumor efficacy of lymphocytes transfer therapy in tumor growth and liver metastasis. (A–D) Growth curves of CT26 and MC38 subcutaneous tumor-bearing mice in different treatment groups, receiving no treatment (“Con”), lymphocyte transfer (“Lym”), LMWH treatment (“Low molecular weight heparin”), or a combination of lymphocyte transfer and LMWH (“L+H”). (B–E) Representative images of CT26 and MC38 subcutaneous tumors in different treatment groups when mice were sacrificed on day 19. (C–F) The tumor weight of CT26 and MC38 subcutaneous tumors in different treatment groups were measured when mice were sacrificed on day 19. (G) Representative images of liver metastasis in each group on day 22. (H) H&E staining of liver metastases (top) and schematic diagram of metastasis identification and tissue typing based on a digital pathological analysis system (bottom). (I) The liver weight in each group was calculated. (J) The liver weight/body weight ratio in each group was calculated. (K) Quantitative analysis of the liver metastasis area/total liver lobe area in each group. LMWH, low molecular weight heparin.
LMWH treatment attenuated hypoxia condition, reduced necrosis, and increased apoptosis in tumors
Hypoxia is a hallmark of solid tumors and correlates with disorganized angiogenesis and poor tissue perfusion.36 Targeted hypoxia reduction could improve intratumoral T-cell function and restore T-cell infiltration, thereby enhancing immunotherapy.37 38 Previous studies have confirmed that LMWH can inhibit pathological angiogenesis by blocking VEGF and other proangiogenic factors.39 40 To investigate whether LMWH reduced the intratumoral heterogeneous blood supply and hypoxic state, we assessed the extent of necrosis and the expression of hypoxia-related markers within the tumor. H&E staining showed that LMWH treatment markedly reduced the necrosis, while more large and complete necrosis was observed in control and only lymphocyte transfer groups, with necrotic regions replaced by complete eosin-staining (online supplemental figure S2A,B). IF analysis of LMWH-treated tumor-bearing mice with Hypoxyprobe revealed that LMWH obviously attenuated overall tumor hypoxia condition (figure 2A). Consistent with H&E and IF staining data, HIF-1α expression also decreased in tumor lesions after LMWH treatment (figure 2B). In addition, TUNEL staining was used to assess the apoptosis of tumor cells. TUNEL analysis showed that the combination therapy significantly increased the apoptosis of tumor cells, while ACT or LMWH treatment alone failed to induce greater cell apoptosis in tumors, as compared with the control group (online supplemental figure S3A,B). These results indicated that LMWH plays an important role in promoting tumor vascular normalization, thereby improving tissue perfusion and reducing hypoxia, and enhancing the antitumor activity of ACT by inducing cancer cell apoptosis.
LMWH treatment attenuated the hypoxia condition within CT26 tumor lesions. (A) Tumor hypoxia was observed with pimonidazole staining (green, Hypoxyprobe-pimonidazole; blue, DAPI). (B) HIF-1α expression in tumor tissues was measured by immunohistochemistry and immunofluorescence. LMWH, low molecular weight heparin.
LMWH had neither cytotoxicity to tumor cells nor ability to activate lymphocytes
Some in vitro experiments were carried out to explore whether the additive antitumor effect of combination therapy was attributable to the cytotoxicity of LMWH itself. It was shown that with the increase of LMWH concentration, LMWH had no direct toxic effect on tumor cells (figure 3A). Subsequently, activated lymphocytes were introduced into the coculture system (effect target ratio=5:1), results showed that only activated lymphocytes, rather than LMWH treatment, had cytotoxicity to tumor cells (figure 3B). Furthermore, the potential of LMWH for the lymphocytes activation was investigated. When LMWH was cocultured with activated or unactivated lymphocytes, the cytotoxicity of lymphocytes towards tumor cells did not show any differences between two groups (figure 3C,D).
LMWH had neither cytotoxicity to tumor cells nor ability to activate lymphocytes. (A) Effects of different concentrations of LMWH on the survival rate of tumor cells. (B) Cytotoxic effect of LMWH combined with or without lymphocytes on tumor cells. (C) Killing effect of LMWH combined with activated/non-activated lymphocytes on tumor cells. (D) Cytotoxic effect of activated/non-activated lymphocytes on tumor cells. (E) Quantitative analysis of CD69 and PD-1 expression on CD8+T cells and CD4+T cells of Balb/c mouse spleen lymphocytes treated with LMWH, concanavalin A, or both for 24 hours. LMWH, low molecular weight heparin.
Previous reports have shown CD69 as a classic marker of the early activation of lymphocytes,41 42 and CD69+CD8+ T cells are considered as the CD8+ cytotoxic T lymphocytes (CTLs) in tumor immunity.43 Moreover, T-cell activation is always accompanied by the upregulation of immune checkpoints such as PD-1 as a brake for immune response and to mediate adaptive immune resistance.44 We then analyzed the expressions of CD69 and PD-1 on lymphocytes treated with LMWH in vitro to determine whether LMWH has an immunomodulatory effect. Although CD4+ and CD8+ T cells in activated groups had greater proportions of them expressing PD-1+ and CD69+ phenotypes compared with unactivated groups, the proportions did not vary in groups combined with LMWH treatment, indicating that LMWH did not affect T cells activation (figure 3E and online supplemental figure S4). Taken together, our results indicate that LMWH treatment neither directly killed nor activated lymphocytes to kill tumor cells.
LMWH enhanced the therapeutic effect of ACT by promoting CD8+ T-cell infiltration
CD8+ T cells play a crucial role in antitumor immunity, and their high density of infiltration in both core and edge of tumors is strongly linked to the positive survival prognosis for patients with CRC.45 Furthermore, PD-1 is rapidly induced on T cells when they shift from a naïve to an activated state in the initial antitumor immunity, thus CD8+PD-1+ T cells are considered as tumor antigen-reactive T cells.46 47We analyzed CD8+ and CD8+PD-1+ T-cell infiltrations in tumors to determine whether the reduced tumor burden in combination therapy was due to their increases. Results showed that the highest numbers of CD8+ and CD8+PD-1+ T cells were detected in both MC38 and CT26 tumors treated with combination therapy, while the amount of CD8+ and CD8+PD-1+ T cells in tumors treated with lymphocyte transfer alone was comparable to that in control tumors (figure 4A–D). Notably, LMWH treatment alone significantly increased the intratumoral CD8+ and CD8+PD-1+ T cells infiltrating in MC38 tumors but not in CT26 tumors (figure 4C,D). This disparity was probably caused by the hypermutated background of MC38 tumors, which allows a stronger infiltration of endogenous CD8+ T cells. In contrast, CT26 tumors with non-hypermutated features exhibit lower immunogenicity, which could only induce limited endogenous CD8+ T cells trafficking into tumors even after LMWH treatment. Our results suggest that LMWH can increase the infiltration of lymphocytes, including CD8+ T cells, into the tumor bed especially when combined with ACT.
LMWH enhanced the therapeutic effect of adoptive cell transfer therapy by promoting CD8+ T-cell infiltration. (A–B) Multiplex immunohistochemistry staining of MC38 and CT26 tumors, CD8 (green), PD-1 (red), DAPI (blue), and CD8+PD-1+ (orange). (C) Quantitative analysis of CD8+ T-cell infiltration. (D) Quantitative analysis of CD8+PD-1+ T-cell infiltration. LMWH, low molecular weight heparin; PD-1, programmed cell death protein 1.
To further determine whether increased CD8+ T-cell infiltration was the potential reason for the enhanced antitumor effect of lymphocyte transfer mediated by LMWH, Balb/c mice were inoculated with CT26 colon cancer cells to establish MSS tumors, and then CD8+ T cells purified from splenic lymphocytes using MACS were transferred via the caudal vein. In vitro activated lymphocytes were labeled with CD8α microbeads and separated from non-CD8+ T cells, then i.p. administration of LMWH and ACT (CD8+ and non-CD8+ T cells) were performed regularly as described in the methods (figure 5A). Results showed that only the combination of CD8+ T-cell transfer and LMWH treatment strongly inhibited tumor growth, while CD8+ T-cell transfer alone induced limited suppression of tumor growth, and other monotherapies and combination therapies showed no significant inhibition on tumor growth versus the control group (figure 5B–D). These findings proved that LMWH can indeed enhance the therapeutic effect of ACT in solid tumors by promoting the infiltration of intratumoral lymphocytes, and this enhanced antitumor immunity was mainly due to the increased CD8+ T cells.
LMWH enhanced the therapeutic effect of adoptive cell transfer therapy by promoting CD8+ T-cell infiltration. (A) Flow chart of the procedure used for LMWH combined with CD8+ T cells/non CD8+ T cells transfer therapy. (B) Representative images of CT26 subcutaneous tumors in different treatment groups when mice were sacrificed on day 19. (C) Growth curves of CT26 subcutaneous tumors in different treatment groups. (D) The tumor weight of CT26 subcutaneous tumors in different treatment groups was measured when mice were sacrificed on day 19. conA, concanavalin A; IL, interleukin; i.p., intraperitoneally; LMWH, low molecular weight heparin; s.c., subcutaneous; WT, wild type.
IVIM in vivo imaging of increased intratumor CD8+ T-cell accumulation by LMWH treatment
To evaluate the capacity of LMWH to promote the infiltration of CD8+ T cells in tumors, we use IVIM in vivo imaging to visualize this process. Balb/c mice inoculated with CT26 cancer cells received twice the pretreatment of LMWH (day 4 and day 7) or not, then activated CD8+ T cells, isolated from splenic lymphocytes by MACS, were labeled with CFSE and transferred into mice through the caudal vein. After CD8+ T-cell transfusion (6 hours, 12 hours, 24 hours, 48 hours), CT26 tumor grafts were imaged by intravital two-photon microscopy to evaluate the migration and infiltration of CD8+ T cells from circulation to the inside of tumors. Tumor blood vessels were indicated by injection of Evans blue through the tail vein before 1 hour at each monitoring time point (figure 6A). When ACT was conducted alone, infused CFSE+CD8+ T cells were scarcely recruited into tumors, and the number of intratumor CFSE+CD8+ T cells had little to no increase within 6–48 hours after transfusion. In contrast, when mice were pretreated with LMWH, the migration and infiltration of CFSE+CD8+ T cells were dramatically increased compared with the only ACT group. The quantification of CFSE+CD8+ T cells in view of each time point was higher, and the number of cells gradually increased overtime and reached a maximum at approximately 48 hours. Moreover, transferred CFSE+CD8+ T cells in tumors with LMWH pretreatment were easier and faster to move out of blood vessel space and infiltrate into tumor stroma as obvious extravasation and perivascular accumulation were observed. As time increased, the increased CFSE+CD8+ T cells disseminated and moved far away from the vasculature and were easier to be observed in the tumor stroma (figure 6B,C and online supplemental video 1–8). Therefore, these findings demonstrate that LMWH enhances the therapeutic effect of ACT by helping the transferred CD8+ T cells infiltrate into solid tumors.
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IVIM in vivo imaging of increased intratumor CD8+T-cell accumulation by LMWH treatment. (A) Flow chart of CFSE-CD8+ T-cell infiltration observed by IVIM in vivo imaging. (B) Representative images of CFSE-CD8+ T-cell infiltration were taken by IVIM in vivo imaging at 6 hours, 12 hours, 24 hours, and 48 hours after cell transfer (blue, angiography Evans blue; green, CFSE-CD8+ T cells). (C) Quantification of CFSE-CD8+ T cells migrating to tumors through blood vessels in live images. conA, concanavalin A; IL, interleukin; CFSE, carboxyfluorescein diacetate succinimidyl ester; i.p., intraperitoneally; LMWH, low molecular weight heparin; WT, wild type.
LMWH-mediated tumor vascular normalization improved the trafficking of CD8+ T cells into tumors
As the concept of “vascular normalization” was laid out, the understanding of the mechanism of antiangiogenic tumor therapy is indeed changing. Vascular normalization highlights that antiangiogenic therapy is not excessively pruning tumor vessels, but producing a morphologically and functionally “normalized” vascular network. In this process, vessels become more mature with reduced vessel diameter and increased pericyte coverage, vessel leakiness is reduced, and immunosuppressive molecules are downregulated on endothelial cells, as a result, the influx of cytotoxic drugs and CTLs are enhanced.36 48 The antiangiogenetic effect of LMWH via proangiogenic factors, such as VEGF and basic fibroblast growth factor (bFGF), has been reported in many studies.39 40 49 To determine whether vascular normalization is the potential reason for increased CD8+ T-cell infiltration mediated by LMWH, we analyzed tumor vascularization and found that LMWH treatment notably decreased the intratumoral level of VEGF-A and reduced the relative CD31+ vascular area in tumors at the termination endpoint (online supplemental figure S5A–D). In addition, to assess vascular maturity, we characterized pericytes by α-SMA+CD31+ immunostaining. As a result, LMWH treatment led to a higher percentage of pericyte-covered blood vessels than in the control group (online supplemental figures S5D,E and S6A). Furthermore, we analyzed the spatial relationship between intratumoral CD8+ T cells and tumor vessels, and found that LMWH significantly promotes the extravasation and perivascular accumulation of CD8+ T cells (online supplemental figure S5C,F). As the aberrant tumor vascular could express Fas ligand (FasL) and establish a selective immune barrier to reject CD8+ T cells homing to tumors, we also analyzed the FasL expression in both ACT and combination therapy. The results indicated that LMWH treatment resulted in a more mature tumor vasculature with decreased FasL expression, thereby promoting more efficient trafficking of CD8+ T cells into the tumor bed (online supplemental figures S6B and S7A–C). These results strongly suggest that LMWH treatment improves the trafficking of CD8+ T cells into tumors, which is probably due to the vascular normalization it mediates.
Combination therapy with LMWH and anti-PD-1 inhibited colon cancer growth and liver metastasis
As we demonstrated that LMWH enhanced the effect of ACT by promoting lymphocyte infiltration in tumors, we wondered if LMWH could enhance the anti-PD-1 efficacy in the same way. Therefore, we tested the therapeutic effect of LMWH combined with anti-PD-1 therapy in the CT26 subcutaneous model. Online supplemental figure S8 shows the in vivo experimental design. As we expected, the combination therapy significantly reduced tumor growth as compared with monotherapy of anti-PD-1 (figure 7A–C). In addition, unlike the MSS tumor of human CRC, which showed almost no response to anti-PD-1 therapy, a relatively obvious therapeutic effect was observed in tumor-bearing mice after receiving only PD-1 blockade (figure 7A–C), which is consistent with the results of previous studies of anti-PD-1 treatment in murine CT26 tumor models.50 51 This result is mainly due to the differences between human and murine tumor cells, specifically, the proportion of TILs expressing PD-1 in murine CT26 tumors is higher than that in human MSS-CRC.52
Combination therapy with LMWH and anti-PD-1 inhibited colon cancer growth and liver metastasis. (A) Representative images of CT26 subcutaneous tumors in different treatment groups when mice were sacrificed on day 19. (B) Growth curves of CT26 subcutaneous tumors in different treatment groups. (C) The tumor weight of CT26 subcutaneous tumors in different treatment groups was measured when mice were sacrificed on day 19. (D) Representative images of liver metastasis in each group on day 22. (E) H&E staining of liver metastases (top) and schematic diagram of metastasis identification and tissue typing based on a digital pathological analysis system (bottom). (F) The liver weight in each group was calculated. (G) The liver weight/body weight ratio in each group was calculated. (H) Quantitative analysis of the liver metastasis area/total liver lobe area in each group. LMWH, low molecular weight heparin; PD-1, programmed cell death protein 1.
Liver is the most common organ for distant metastasis of CRC,53 and liver metastasis is the main cause of CRC cancer-related death.54 Unlike primary MSS-CRC, many clinical studies in metastatic MSS-CRC suggested that the combination strategy of ICIs and antiangiogenic agents presented a low or no response in patients with liver metastasis.29–32 Therefore, we investigated the efficacy of LMWH combined with anti-PD-1 therapy in CRC liver metastasis. Our results showed that LMWH combined with PD-1 blocking dramatically reduced liver metastasis in mice (figure 7D–E), and the corresponding liver weight, liver weight to body weight, and the rate of metastasis area to total liver area were all significantly reduced (figure 7F–H). However, as same as ACT therapy, PD-1 therapy alone had no significant therapeutic effect on liver metastasis. This is mainly because the liver has a unique “local immune tolerance” characterization,55 which tends to develop a highly suppressive immune microenvironment when metastasis occurs, to promote tumor growth and restrain the efficacy of immunotherapy.56 Our findings suggest that, via the vascular normalization, LMWH promotes more anti-PD-1 antibody and CTLs delivered into tumors, reverses the suppressive immune microenvironment, and thereby enhances immunotherapy efficacy.
LMWH treatment did not directly modify CD8+ T-cell function but modified the TME favoring chemoattraction to enhance immunotherapy efficacy
To determine whether LMWH has a direct modification of intratumoral CD8+ T-cell function, we measured the expression of Ki67 and granzyme-B (GZMB) in CD8+ T cells as indicators of proliferation and cytotoxicity of CTLs, respectively. Our results showed that LMWH treatment alone had no impact on the infiltration of CD8+Ki67+ T cells and CD8+GZMB+ T cells (“IgG2a” vs “IgG2a+LMWH”). The anti-PD-1 treatment alone moderately increased the infiltration of CD8+Ki67+ T cells and CD8+GZMB+ T cells. Interestingly, the combination therapy of anti-PD-1 and LMWH significantly increased the infiltration of CD8+Ki67+ T cells and CD8+GZMB+ T cells (figure 8). This result indicated that LMWH treatment did not directly modify CD8+ T-cell function but induced a “vascular normalized” TME allowing more efficient delivery of CD8+ T cells and anti-PD-1 antibody, thereby reversing the “suppressed” and “exhausted” CD8+ T cells to “cytotoxic” and “proliferative” CTLs and enhancing the immunotherapy.
LMWH treatment improved the infiltration of proliferative and cytotoxic CD8+ T cells mediated by anti-PD-1 therapy. (A) mIHC staining of CT26 tumors, CD8 (green), Ki67 (red), DAPI (blue). (B) mIHC staining of CT26 tumors, CD8 (red), GZMB (green), DAPI (blue). (C) Quantitative analysis of CD8+Ki67+ T-cell infiltration. (D) Quantitative analysis of CD8+GZMB+ T-cell infiltration. GZMB, granzyme-B; mIHC, multiplex immunohistochemistry; LMWH, low molecular weight heparin; PD-1, programmed cell death protein 1.
Trafficking CTLs into solid tumors is a tightly controlled process by chemokines and chemokine-receptors.48 In CRC, the upregulated expression of the chemokine CXLC10 is well correlated with increased CTLs infiltration.57–59 We treated CT26 tumor-bearing mice with LMWH and found a higher expression of CXCL10 in tumors as compared with that in the control group (online supplemental figure S9A,B). Given that CXCL10 expression is regulated by interferon (INF)-γ signaling,60 and the inhibition of VEGF/VEGF receptor (VEGFR) and bFGF/VEGFR signaling pathways and reduction of tumor hypoxia could reactivate the IFN-γ signaling,61–63 a reasonable explanation could be proposed that LMWH inhibited VEGF and bFGF, normalized tumor vasculature, and reduced intratumoral hypoxia, thereby restoring the IFN-γ production and increasing the CXCL10 expression.
The CXCR3-CXCL10 axis is mainly responsible for the recruitment of CD8+ T cells into tumors.64 We further evaluated the CXCR3 expression in each group to determine whether LMWH-mediated synergism in anti-PD-1 therapy is dependent on the CXCR3 system. As we expected, anti-PD-1 therapy induced an increase of CD8+CXCR3+ T cells in tumors, and its combination with LMWH treatment further increased the infiltration of CD8+CXCR3+ T cells (online supplemental figure S10). This result is consistent with the therapeutic data of anti-PD-1 in murine CT26 subcutaneous and liver metastasis models (figure 7), demonstrating a TME that favors chemoattraction has been modified and benefits the antitumor response. However, CD8+ T cells in tumors treated with LMWH alone had no significant increase in CXCR3 expression, suggesting LMWH has no direct effect on CXCR3 expression. LMWH treatment induced more normalized tumor vascular with better maturity, lower expression of FasL, higher expression of CXCL10, and more efficient delivery of CD8+ T cells and PD-1 antibody into tumors, and CD8+CXCR3+ T cells were recruited and trafficked more easily into tumors, thereby, providing anti-PD-1 therapy with more adequate “substrates” to work with in the tumor, which was reversed from “cold” to “hot”.
Discussion
Delivering more tumor-responsive T cells into the TME or reversing T-cell suppression and exhaustion in the TME is an critical step in CRC antitumor immunity response. However, for the majority of the CRC population, ACT and ICIs show only limited clinical response, and the therapeutic benefit is lower in patients with liver metastases.6 27 31 65 66 It is probably due to the “barrier” formed by aberrant tumor vasculature, the immune escape caused by a lack of immunogenicity in most CRC tumors (MSS type), or a combination of both, resulting in tumors that lack immune infiltration and are insensitive to immunotherapy. It is necessary to explore new strategies to improve the immune infiltration of CRC tumors and enhance the efficacy of immunotherapy.
Our results showed that LMWH promoted lymphocyte infiltration in tumors. Despite the fact that monotherapy of LMWH was ineffective, it significantly increased the tumor sensitivity to adoptive and anti-PD-1-based immunotherapies, resulting in the synergistic antitumor effect. LMWH plus ACT significantly enhanced the antitumor immune response in both “immune-excluded” CT26 tumors and “immune-inflamed” MC38 tumors via vascular normalization. Abnormal tumor blood vessels can restrict lymphocytes trafficking into tumors in various ways, such as by inhibiting of the dentritic cell maturation and antigen presentation, and T cells development, thereby forming an immunosuppressive TME.67 68 Additionally, reduced aggregation of adhesion molecules on tumor endothelial cells could inhibit lymphocytes adhesion to tumor vessels,69 and endothelial cells may specifically express FasL to recruit T regular cells and reject CD8+ T cells migrating into tumors.12 Moreover, reduced pericyte-coverage around the tumor vasculature leads to increased vascular permeability and decreased hydrostatic pressure, resulting in poor tissue perfusion and impairing lymphocytes infiltration into tumors.10 A large number of studies have reported that LMWH exerts an antiangiogenic effect by antagonizing various growth factors, such as VEGF and bFGF.39 70 Therefore, we confirmed that LMWH promoted the normalization of tumor vessels, relieved intratumoral hypoxia, and promoted the infiltration of lymphocytes, including CD8+ T cells, into tumors, thus enhancing the therapeutic effect of ACT. Furthermore, by transferring CD8+ versus non-CD8+ T cells, we confirmed that the antitumor effect is indeed mediated by increased CD8+ T-cell infiltration in the combination therapy of LMWH plus ACT.
There was no significant antitumor effect when LMWH was used alone. This result is consistent with previous reports that LMWH does not inhibit tumor growth in situ.35 70 On the one hand, the reduced necrosis and hypoxic state in tumors after LMWH treatment could promote tumor growth. On the other hand, the normalized tumor vasculature after LMWH treatment could allow better CD8+ T-cell infiltration to inhibit tumor growth. Therefore, the antitumor synergism in the LMWH plus ACT strategy is not simply the sum of the effects of these two monotherapies, but the normalized tumor vasculature and the modified TME help T cells to infiltrate into tumor and thereby mediating a better antitumor response. Many studies of antiangiogenic drugs have shown similar results, that is anti-VEGF monotherapy caused limited or even no tumor regression, but evidence of underlying vascular normalization was observed.24 71 72 Given that this vascular-immune crosstalk mediated by LMWH, we further assessed the efficacy of LMWH combined with anti-PD-1 therapy. As we expected, the results showed that LMWH plus PD-1 blocking significantly inhibited the tumor growth. Many preclinical studies have reported the antimetastatic effects of LMWH, including the inhibition of local invasion by preventing heparase mediated ECM degradation,20 73 and the inhibition of cancer cells adhesion to platelets or endothelial cells in heparin-mediated circulation.74 75 In our study, LMWH treatment alone did not show any significant antimetastatic effect. However, when LMWH was combined with ACT or anti-PD-1 therapy, it significantly reduced the development of liver metastasis, suggesting the synergistic antitumor effect in liver metastasis was also associated with the antitumoral vascular-immune crosstalk after LMWH administration, but not the simply additive antimetastatic effects of LMWH reported previously.
Although the strategy of antiangiogenic agents combined with ICIs has been reported to induce an immune-stimulatory effect to MSS-CRC in some preclinical and clinical studies, this strategy is still being tested in clinical trials, and some problems cannot be ignored. First, the use of antiangiogenic agents, ICIs, and both of them may increase the risk of cancer-associated venous thromboembolism (CAT).76–79 Second, recent clinical trials have demonstrated the incidence of grade ≥3 treatment-related adverse events of combination therapy (anti-PD-1 plus antiangiogenesis), such as palmar plantar erythrodysesthesia syndrome, hypertension, proteinuria and elevated transaminase, occurred in 30–50% of patients, leading to dose interruptions and reductions and impairing the therapeutic effects.30 32 80 81 Third, patients with liver metastases have an obviously lower objective response rate than those without liver metastases in clinical trials, indicating that the MSS-CRC with liver metastases is not sensitive to this combination strategy. Totally, as compared with present antiangiogenic agents, LMWH has been widely used in the prevention and control of CAT during the process of antiangiogenic therapy and ICIs therapy for its anticoagulant characteristics, and is less toxic and less expensive, and the simple combination therapy achieves improved immunotherapy effectiveness even in the liver metastatic model.
Interestingly, two clinical trials of LMWH combined with standard treatment in patients with small cell lung cancer reported paradoxical results. In a study by Lebeau et al, patients receiving heparin treatment obtained better clinical response and survival,22 however, a study by Ek et al (RASTEN trial) reported a negative outcome.82 We found that patients in both studies were given a supraprophylactic dose of LMWH, which is reasonable and necessary, because only a higher concentration may compete with ligand interactions and downstream functional effects, such as angiogenesis and metastasis.83 However, the dose of heparin treated in the RASTEN trial was still significantly lower than that in study by Lebeau et al. Therefore, we propose that there was probably no satisfactory inhibition of proangiogenic factors, and no vascular normalization was induced in tumors in the RASTEN study, thereby chemical drugs could not be better delivered into tumors. Despite the supraprophylactic dose of LMWH is related to underlying hemorrhagic events, the significant clinical implications of combination therapy with LMWH and immunotherapy should not be ignored. Many LMWH derivatives have been reported as the “non-anticoagulant heparin” after chemical modification, and LMWH obtained a lower anticoagulant function and a higher affinity to growth factors.84–86 Thus, our findings provided a strong support for the development of non-anticoagulant heparin for the combination strategy (antiangiogenesis plus immunotherapy), and for solving current problems in the clinical application of antiangiogenic drugs.
In conclusion, our study highlights that combined LMWH treatment promoted lymphocyte infiltration and enhanced the efficacy of adoptive and anti-PD-1-based immunotherapy in murine CRC models. Since LMWH does not cause side effects similar to current antiangiogenic agents, and its good anticoagulant properties can prevent the cancer-related hypercoagulant state and CAT, as well as its better reduction of liver metastasis when combined with immunotherapy, the findings support further translational research and clinical trials for CRC, especially the “cold” MSS-CRC tumors.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
All animal studies were reviewed and approved by the Ethics Committee for Animal Experiments of South China University of Technology (AEC:2022066).
Acknowledgments
Flow charts were created with BioRender.com.
References
Supplementary materials
Supplementary Data
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Footnotes
YQ, JH, QZ and LZ contributed equally.
Contributors FW, YQ, JH, KX and WL designed all the experiments and wrote the manuscript, YQ, QZ, LZ, QS and HH performed the experiments, and FW and YQ also collected and analyzed the data, contributed to the writing of the manuscript and reviewed and/or edited the manuscript before its submission. FW also made substantial contributions to the discussions of the content. FW is responsible for the overall content and is the guarantor of this manuscript. All authors reviewed and approved the manuscript.
Funding The work is partly supported by National Natural Science Foundation of China (#81972780), Guangzhou Municipality Bureau of Science and Technology, Guangzhou, China (#202201020521, #202102010033, #202201020281, #202002030288) and Natural Science Foundation of Guangdong Province (#2020A1515010051).
Competing interests Authors declare no conflict of interest.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.