A Practical Spatial Analysis Method for Elucidating the Biological Mechanisms of Disseminated Cancers in Vivo

doi:10.21203/rs.3.rs-1444820/v1

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A Practical Spatial Analysis Method for Elucidating the Biological Mechanisms of Disseminated Cancers in Vivo

https://doi.org/10.21203/rs.3.rs-1444820/v1

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The elucidation of spatial interactions between cancer and host cells is important for the development of new therapies against disseminated cancers. In this study, we developed a practical spatial analysis method using a gel-based embedding system and applied it to a murine model of cancer dissemination. After euthanization, every abdominal organ enclosed in the peritoneum was extracted en bloc. We injected agarose gel into the peritoneal cavities to preserve the spatial locations of organs including metastatic niches, then produced specimens after the gel was solidified. Preservation of the original spatial localization was confirmed by comparing the magnetic resonance imaging results to the sectioned specimens. We examined the effects of spatial localization on cancer hypoxia using hypoxia markers immunohistochemically. Finally, we identified the mRNA expression on the specimens and demonstrated the applicability of spatial genetic analysis. In conclusion, we established a practical method for the in vivo investigation of spatial location-specific biological mechanisms on disseminated cancers.

gel-based specimen preparation method

spatial analysis

cancer dissemination

cancer metastasis

hypoxia

lipid metabolism

Several biological mechanisms, including those involving epigenetically regulated signal transductions, occur only at specific spatial locations[1–4]. In cancer research, spatial cell–cell interactions are important at every step of cancer progression including metastasis and dissemination. For example, spatial transcriptome analysis has shown that the lymphoid area close to the tumor region specifically expresses cancer-promoting genes compared to the lymphoid area away from the tumor area[4]. Moreover, spatial tissue analysis has shown that cancer cells promote remodeling of the tumor microenvironment via transforming growth factor beta (TGFβ signaling[5]. Therefore, spatial analysis is essential for understanding the precise interactions between cancer cells and the surrounding host cells especially in the metastasized or disseminated microenvironments.

Spatial information-based analysis requires tools that provide detailed anatomical information. Currently, spatial information is obtained from imaging techniques such as computed tomography, magnetic resonance imaging (MRI), and ultrasound scanning. These imaging systems are used to assess the extent of tumor spread and metastasis for clinical staging and treatment selection[6–8]. Although these tools provide relevant spatial information, whenever it is necessary to examine detailed spatial location-specific biological mechanisms in the tumor microenvironment, other tools that can directly access tumor tissues are required.

Pathological specimens possess spatial information as well as biological information. Recently, effective genetic spatial analysis tools have become commercially available and are applied to histopathological specimens[9–11]. In general, histopathological analysis requires the extraction and dissection of each organ separately without preserving any spatial information. Although some methods of preserving spatial information have been proposed, they are inadequate for the precise preservation of spatial information[12, 13].

Herein, we developed a novel and simple method for the preparation of specimens intended for spatial biological investigations in mice that involves solidifying all abdominal organs together using agarose gel. By focusing on disseminated ovarian cancer, we demonstrated the utility of our system by using it to investigate two spatial location-dependent biological mechanisms—i.e., hypoxia and lipid metabolism. We also showed that RNA is preserved from in situ hybridization using the method, thereby demonstrating its applicability to spatial genetic and epigenetic analyses. Our model provides details on the microscopic structure and immunophenotype of the tissue under scrutiny, while preserving complex spatial information including metastatic niches. Moreover, it can reveal the previously undetectable biological mechanisms of the microenvironments and meet the needs of any research that requires in vivo spatial information.

Cell lines and cell culture

ES-2 (CRL-1978) was purchased from ATCC (Manassas, Virginia, USA), and RMG-1 (JCRB0172) was purchased from JCRB Cell Bank (Osaka, Japan). These cell lines are derived from human ovarian clear cell carcinoma and are often used in mouse xenograft models[14, 15]. Cells were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in a humidified atmosphere comprising 5% CO₂. Semi-confluent cells were dissociated using 0.05% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA). Centrifugation was performed for 3 min at 180 × g at room temperature to obtain a cell pellet.

Cancer Dissemination Model

All animal studies were approved by the Institutional Animal Experiment Committee (Approval No. 01–07). Female BALB/c-nu/nu mice aged 6–8 weeks (Charles River Japan, Yokohama, Japan) were used for the experiments. We injected either 1 × 10⁶ ES-2 cells or 1 × 10⁷ RMG-1 cells intraperitoneally. The tumor-injected mice were sacrificed by cervical dislocation following anesthesia after 2 weeks (ES-2) or 5 weeks (RMG-1). Except supplementary information, we used ES-2 in the present study because of the rapid growth and massive dissemination phenotype. For the hypoxia study, a HypoxyprobeTM-1 solution (Hypoxyprobe Inc., Burlington, MA, USA) in saline was injected intraperitoneally into the mice at a dosage of 60 mg/kg, 30 minutes before sacrifice.

Spatial Sectioning Method

The workflow for preparing the specimens is shown in Fig. 1a. Disseminated cancer with a few ascites developed either two weeks (in the case of the ES-2 cells) or five weeks (in the case of the RMG-1 cells) after injecting the cancer cells into the peritoneal cavities of the mice. The mice were euthanized under general anesthesia at that time-point. The abdominal organs enclosed in the parietal peritoneum—including the erector spinae muscles, the vertebrae, and the skin—were extracted en bloc. Then, 1–2 ml of 10% neutral buffered formalin was injected into each peritoneal cavity. Thereafter, the organs in the parietal peritoneum were further soaked in 10% neutral buffered formalin for 1 day in a plastic tube. Subsequently, the 10% neutral buffered formalin in both the peritoneal cavity and the tube was replaced with neutral EDTA to decalcify the vertebrae. After 1 week, the EDTA in the peritoneal cavity was replaced with 1–2 ml of 3–10% agarose gel (#E-3120-500 Gene Pure LE, ISC BioExpress, Kaysville, Utah, USA), and the organs in the parietal peritoneum were soaked in a 50 ml tube filled with 10% agarose gel. The tube was stored at room temperature (24–26°C) until the gel hardened. The organs in the parietal peritoneum were carefully removed from the tube and serially sliced into uniform 3-mm-thick sections. The sliced organs were then embedded in paraffin and sectioned for histopathological analysis. We also utilized another method involving abdominal organs extracted en bloc without the parietal peritoneum (Fig. S1).

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

Each formalin-fixed paraffin-embedded tissue specimen (4-µm thick) was placed on a glass slide and stained with H&E. The deparaffinized and rehydrated slides were immersed in 0.01 M citrate at pH 6.0 (Sigma-Aldrich, St. Louis, Missouri, USA), and heat-induced antigen retrieval was performed in an autoclave at 110°C for 15 min. The slides were put on the tabletop to cool to room temperature, washed in phosphate-buffered saline, and immersed in 3% H₂O₂ diluted with methanol. Background Sniper (Biocare Medical, Pacheco, CA, USA) was used for blocking as required. Primary antibodies against hypoxia-inducible factor-α (HIF1α) (EP1215Y, diluted at 1:100, rabbit monoclonal IgG; Abcam, Cambridge, UK), hypoxyprobe-1 (PAb2627AP, diluted at 1: 250, rabbit polyclonal IgG antibody; Hypoxyprobe Inc., Burlington, MA, USA), CD31 (D8V9E, diluted at 1:100, rabbit monoclonal IgG; Cell Signaling Technology, Danvers, MA, USA), UCP1 (ab234430, diluted at 1:100, rabbit monoclonal IgG; Abcam), FABP4 (ab92501, diluted at 1:100, rabbit monoclonal IgG; Abcam), CD36 (ab252923, diluted at 1:100, rabbit monoclonal IgG; Abcam), fatty acid synthase (FASN; 3180S, diluted at 1:100, rabbit monoclonal IgG; Cell Signaling Technology), and human leukocyte antigen (HLA; ab52922, diluted at 1:250, rabbit monoclonal IgG; Abcam) were used for IHC in the present study. Histofine® high stain PO (MULTI) (Nichirei, Tokyo, Japan) or Histofine® simple stain mouse MAX-PO (R) (Nichirei) and a Histofine® DAB substrate kit (Nichirei) were used to detect the labeled antigens. Non-specific mouse or rabbit IgG was used as a negative control. We used ImageJ software (version 1.52a)[16] to analyze the IHC slides.

RNA- in situ hybridization (RNA-ISH)

We performed RNA-ISH on the formalin-fixed paraffin-embedded slides. An RNAscope™ kit (Advanced Cell Diagnostics, Hayward, CA, USA) was used for RNA-ISH. RNA-ISH was performed according to manufacturer’s protocol. mRNA expression in the specimens was detected using a negative control probe, positive control probe and anti-FABP4 probe (310043, 313901 and 470641-C2, Advanced Cell Diagnostics).

Acquisition Of Histological Images

The specimen slides were scanned with an Aperio SC2 scanner (Leica Biosystems, Wetzlar, Germany) and imaged with an Aperio eSlide Manager (Leica Biosystems). The sections were digitally imaged at 20× magnification and saved as TIFF files.

Evaluation Of Immunohistochemical Staining

Vascular regions (i.e., tumor areas within 100 µm of CD31-positive vessels) and avascular regions (i.e., tumor area 100 µm away from CD31-positive vessels) that stained positive for HIF1α and hyopoxyprobe-1 were analyzed using Image J software (Version 1.52a)[16]. Briefly, we took 26 color images from randomly selected vascular or avascular areas in the stained specimens at 20× magnification. The non-tumor regions and necrotic regions were removed. The color images were converted to 8-bit grayscale, and an appropriate threshold was set prior to Image J analysis.

Evaluation Of The Accuracy Of Our System For Preserving Spatial Information By Mri Imaging (Mri)

We performed MRI to evaluate the accuracy of the anatomical information obtained and preserved using our method at the Central Institute for Experimental Animals (Yokohama, Japan). The mice were anesthetized with 1.5% isoflurane. Respiration and rectal temperature were monitored during the measurements. MRI was performed using a 7.0 tesla MRI system equipped with actively shielded gradients at a maximum strength of 700 mT/m (BioSpec 70/16; Bruker BioSpin GmbH, Ettlingen, Germany) and with a 38 mm volume coil. T2-weighted images of the axial plane were acquired using a rapid acquisition with relaxation enhancement method with the following parameters: effective echo time (TE), 40 ms; repetition time (TR), 3500 ms; rapid acquisition with relaxation enhancement factor, 8; number of averages, 4; spatial resolution, 150 × 150 µm; slice thickness, 0.75 mm; and number of slices, 36.

Statistical analysis

In the present study, statistical analysis was performed using SPSS Statistics version 19 (IBM, Armonk, NY, USA). The statistical significance was determined using an unpaired t-test. The variables were compared using Pearson’s correlation coefficient. A P-value of < 0.05 was considered statistically significant.

Establishment of the gel embedding method that preserves spatial information

To develop a multiorgan evaluation system that preserves spatial information, we employed an agarose gel-embedding method. In the present study, we applied this system to a murine model of ovarian cancer dissemination because of the importance of three-dimensional interactions in cancer dissemination and therapeutic resistance[17, 18]. We injected cells of either of the well-known ovarian cancer cell lines ES-2 or RMG-1 into the abdomens of mice to create an ovarian dissemination model[19, 20]. Except supplementary information, we used ES-2 in the present study due to the rapid growth and massive dissemination phenotype from preliminary studies. After 2–5 weeks, the cancer had spread, and ascites had developed. The organs in the parietal peritoneum were solidified with 10% agarose gel after fixation and decalcification (Fig. 1a). The spatial location of each solidified organ was thereby preserved, which facilitated equidistant serial sectioning. As shown in Fig. 1b, multiple organs in their original locations were observed in each section. In the represented slide, the intestines (I), kidneys (K), pancreas (P), liver (L), and spleen (S) are visible in the same section (Fig. 1b). In addition, as shown in Fig. 1c, complicated metastatic niches were preserved in the specimens. At the pelvic peritoneal cavity, metastatic niche formed by disseminated cancer cells, retroperitoneal adipose tissues, and periovarian adipose tissues was observed (Fig. 1c, left two images). Also, at the upper peritoneal cavity, another metastatic niche formed by disseminated cancer cells, pancreatic tissue, pancreatic duct, major omentum, and stomach was observed (Fig. 1c, right two images). From these results, we successfully established an in vivo specimen preparation method for spatial analysis.

Evaluation Of The Accuracy Of Our System For Preserving Spatial Information By Mri

To evaluate the accuracy and reproducibility of our system for obtaining abdominal spatial information, we applied MRI to our dissemination model. We imaged the disseminated cancer in the model mice and prepared sections from the mice after imaging. Using our method, the positions of the solid organs as determined using MRI, were preserved in the macro specimens and in the H&E-stained sections (Fig. 2a, 2b). Meanwhile, the hollow organs were mobile in the peritoneal cavity, therefore the positions of these organs were changed (Fig. 2a, 2b). Sequential horizontal images obtained using MRI from the mice confirmed that the spatial information of solid organs was preserved throughout the peritoneal cavity in the sectioned tissues (Fig. 2b). Therefore, MRI confirmed the reproducibility of the spatial information obtained using our system. Histopathological analysis also allowed us to detect very small tumor nodules that evaded detection using MRI (Fig. 2c). Thus, our model allows for a detailed analysis of lesions while preserving spatial information.

Functional Analysis: Preserving Spatial Information Pertaining To Hypoxia In The Tumor Environment

Next, we attempted to determine the potential usefulness of our method for elucidating biological functions at the metastatic niche only specifically observed in our method. In general, although severe hypoxia is associated with metastasized cancer, the exact extent of the hypoxia is unclear[21]. Determining the degree of hypoxia associated with disseminated cancers may facilitate the development of effective therapies. We performed IHC analysis of HIF1α and hypoxyprobe-1 (pimonidazole derivatives), which are hypoxia markers[22, 23], to quantify the hypoxia associated with the cancer cells. As shown in Fig. 3a, we measured hypoxia at the the metastatic niche composed by disseminated cancer cells, the retroperitoneal connective tissue, uterus, and periovarian visceral adipose tissue shown in Fig. 1, and it was possible to visualize the disseminated ovarian cancer cells using anti-HLA staining. We compared the extent of HIF1α and hypoxyprobe-1 staining in the vascular and avascular areas. Both HIF1α and hypoxyprobe-1 staining were significantly more prevalent in the avascular regions than in the vascular regions (Fig. 3b). There was a significant correlation between the staining patterns of HIF1α and hypoxyprobe-1 in the same cases (Fig. 3c). Interestingly, there was a greater degree of hypoxyprobe-1 staining in the cancer cells floating in the peritoneal cavity than in the intra-abdominal cancer cells supplied by numerous CD31-positive vessels (Fig. 3a and Fig. 3d). These results suggest that our model can be used to accurately assess the level of hypoxia in spatially disseminated abdominal tumors. Next, we attempted to determine the metastatic niche-specific metabolic processes. Lipid metabolism is facilitated by the hypoxic conditions in cancer cells[24, 25]. Furthermore, in the peritoneal cavity environment, visceral adipocytes, which provide lipid droplets, are major host cells that interact with disseminated cancer[26, 27]. We used FASN staining to investigate lipid metabolism in the cancer cells and found that the disseminated cancer cells stained positive for FASN (Fig. 4a) at the metastatic niche same as Fig. 3. CD36 is a key molecule in fatty acid transport which generally expresses in both white and brown adipocytes; the disseminated cancer cells stained slightly for CD36 (Fig. 4a). Moreover, it has been reported that white adipocytes positively support tumor development[28, 29]. In accordance with those reports, the disseminated cancer cells mainly attached to and infiltrated UCP1(a marker for brown adipocytes) negative and Perillipin1 (a marker for pan-adipocytes) positive white adipocytes at the metastatic niche (Fig. 4a).

Potential Analysis Of Spatial Gene Expression

Finally, we assessed the applicability of our system to the latest spatial genetic analysis techniques[11, 30] by performing RNA-ISH on the specimens. As indicated in red in Fig. 4b, there was significant human mRNA expression in the disseminated human cancer cells (red colored area) that infiltrated the greater omentum and pancreas at the metastatic niche. Also, specific mRNA expression of lipid transport-specific gene (FABP4) was also observed in the retroperitoneal adipose tissue (Fig. S2). These results suggest that spatial mRNA expression analysis can be performed on specimens using our method.

We established a novel method for comprehensively accessing detailed spatial information pertaining to biological mechanisms in the peritoneum. Our method can be used to evaluate the volume of disseminated tumors and investigate spatial location-specific cancer development mechanisms. To date, evaluation of the volume of disseminated tumors has mainly been based on the macroscopic examination of resected specimens or in vivo fluorescence imaging[31–33]. Our method offers definite advantages over those two techniques; compared to macroscopic examination, our approach enables screening of the entire peritoneal cavity without overlooking small tumor nodules and does not require consideration of attenuation or irregular fluorescence due to the imaging conditions. Furthermore, only our method enables the examination of inter-multiple organ niche-specific biological mechanisms. Disseminated tumor masses frequently interact with multiple organs. Thus, our gel-embedding method—which preserves spatial information pertaining to multiple organs—can be used to unravel the niche-specific mechanisms of disseminated cancer.

We evaluated the accuracy of the spatial information obtained using our method by MRI. As expected, the position of every organ according to MRI corresponded to that observed in the sectioned tissue processed by our method, which preserved the spatial information pertaining to all the abdominal organs. However, our method involves filling the peritoneal cavity with agarose gel, which changes the positions of the greater omentum and the cancer cells floating in the ascites. Therefore, our method is most suited to the evaluation of the spatial location-specific biological mechanisms of organs that are fixed to the parietal peritoneum.

Figure 3 and Fig. 4 demonstrate the potential of our method as a tool for the examination of biological mechanisms. In this regard, oxygen concentration in cancer cells has been investigated[34–36]. However, the role of hypoxia in disseminated intraperitoneal cancer remains unclear. In the present study, we divided the tumors into vascular and avascular regions based on a previous report[37]. We confirmed the hypoxic status of the tissues using two major hypoxia markers, HIF1α and hypoxyprobe-1. Under hypoxic conditions, HIF1α is an important transcription factor, and its stability is regulated by the oxygen-dependent prolyl hydroxylase domain protein and von Hippel–Lindau tumor suppressor protein axis[38, 39]. Pimonidazole, which is a 2-nitroimidazole adduct, has often been used as a sensitive hypoxic marker since Varghese et al. reported its use in hypoxic cells, both in vitro and in vivo [36, 40]. Hypoxyprobe-1 (pimonidazole hydrochloride) can detect the adducts formed under hypoxic conditions (pO₂ < 10 mmHg) and has been widely used, both in vitro and in vivo[41–43]. As shown in Fig. 3, the staining intensities due to both HIF1α and hypoxyprobe-1 were higher in the avascular region than in the vascular region. In addition, confirming previously reported results[44], the staining area due to hypoxyprobe-1, which reflects “chronic hypoxia”, was smaller than that due to HIF1α in the avascular area. Floating cancer cells in ascites were highly expressed hypoxyprobe-1 compared to disseminated cells. These results suggest that our model can elucidate hypoxic conditions of disseminated cancer at the metastatic niches. Lipid metabolism in cancer cells is closely related to hypoxia[24, 25]. Free fatty acids act as nutrients for cancer cells under hypoxic conditions, and white adipocytes continuously release fatty acids via transporters. As shown in Fig. 4, using IHC, we investigated lipid metabolism and fatty acid intake at the metastatic niche composed by the retroperitoneal connective tissue including brown adipose tissue, uterus, and periovarian visceral adipose tissue. The cancer cells disseminated in these white adipose tissues express FASN, which is a marker of lipid metabolism, and CD36, which is a transporter of extrinsic fatty acids. Our results suggest that, in addition to hypoxia, adipose tissue may favor disseminated/metastasized cancer cells in the peritoneal cavity. We could show that our method is able to examine spatial location-specific protein expression at the metastatic niche composed of multiple organs that has not been previously detected using other methods.

Finally, we confirmed the applicability of our method to spatial genetic expression analysis. Recently, several novel methods have been developed for spatial genetic analysis[11, 30, 45]. We performed RNA-ISH on the specimens, thereby demonstrating that our method preserves mRNA in the disseminated cancer in multiple organs and is appropriate for spatial genetic expression analysis.

Although cancer dissemination is difficult to quantify, attempts have been made to evaluate tumor masses disseminated in the peritoneal cavity[13, 46, 47]. The peritoneal cancer index (PCI) method is the most frequently used technique for assessing cancer dissemination[46, 48, 49]. To evaluate cancer dissemination, the PCI method involves categorization of the abdominal area into nine sections and counting of visually confirmed disseminated nodules in each of those sections (Supplementary Fig. 3). In contrast, our method simultaneously evaluates the whole peritoneal cavity and the peritoneum, using IHC staining for human-derived tumor-specific proteins, such as HLA (Supplementary Fig. 3). The PCI and our method offer different advantages and disadvantages. Compared with the PCI method, our approach preserves spatial information and may evaluate more precise and objective tumor area. However, our method has several limitations. The intraperitoneal organs solidified in agarose gel must be sliced into sections that are at least 3-mm thick. Therefore, depending on the thickness of the slice, some organs or lesions may be overlooked. Nevertheless, our method still offers advantages for the quantitative analysis of cancer dissemination compared to the PCI method, which reportedly yields differences between the scores in the traditional macroscopic PCI and the pathologically evaluated PCI[50]. Another limitation of the present study is that we used a decalcifying reagent (EDTA) in addition to formalin to prepare the specimens. Such reagents reduce the sustainability of proteins and nucleic acids compared to those in fresh tissue. Our method requires further improvement for the detailed investigation of proteins and genetic analyses, such as the sectioning of frozen non-fixed and non-decalcified samples.

In conclusion, we established a practical method that preserves the spatial information of metastatic niches pertaining to multiple organs. We also demonstrated the usefulness of our method for investigating hypoxic conditions and lipid metabolism and indicated its potential for spatial genetic analysis. Our method is suitable for investigation of the biological mechanisms involved in spatial cell–cell, tissue–tissue, and organ–organ interactions in the peritoneal cavity.

Acknowledgments

We appreciate the support provided for the MRI study by Yuji Komaki, Chihoko Yamada (Central Institute for Experimental Animals), and KensakuAita (In-Vivo Science Inc.). We would also like to thank Kumiko Ohrui for helping us with the in vivo experiments.

Ethics Approval/Consent to Participate Statement

No data involving human patients or tissue were included and therefore our study did not require ethical approval for human rights. All animal protocols had ethical approval from the Kanagawa Cancer Center Institutional Animal Experiment Committee (Approval No. 01-07).

Data Availability Statement

Data generated as part of this study but not presented in the manuscript

or supplementary material are available from the corresponding author upon reasonable request.

Author Contribution Statement

S.S. performed study concept, experimental design, interpretation of results, writing, review, and revision of the paper; Y.O. performed all experiments, interpretation of results, writing, and improving specimen preparation protocols; Y.M. and E.M. supervised this study. M.Y. and Y.N. performed preparation of specimens and all staining.

Statements and Declarations

The authors have no conflicts of interest to declare.

Funding Statement

This work was supported by JSPS Grants-in-Aid for Scientific Research (KAKENHI; grant number JP20K09422 to Shinya Sato), and by the Takeda Science Foundation (to Shinya Sato). Figures are cited from Biorender.com.

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No competing interests reported.

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A Practical Spatial Analysis Method for Elucidating the Biological Mechanisms of Disseminated Cancers in Vivo

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Abstract

Figures

Introduction

Materials And Methods

Cell lines and cell culture

Cancer Dissemination Model

Spatial Sectioning Method

Acquisition Of Histological Images

Evaluation Of Immunohistochemical Staining

Evaluation Of The Accuracy Of Our System For Preserving Spatial Information By Mri Imaging (Mri)

Statistical analysis

Results

Establishment of the gel embedding method that preserves spatial information

Evaluation Of The Accuracy Of Our System For Preserving Spatial Information By Mri

Functional Analysis: Preserving Spatial Information Pertaining To Hypoxia In The Tumor Environment

Potential Analysis Of Spatial Gene Expression

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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