Abstract

   Ascites and pleural effusion are recognized complications of pancreatic
   cancer. These diseases are accompanied by ascites and pleural effusion,
   and drug treatment is limited by high costs, long hospital stays, and
   failure rates. Immunotherapy may offer new option, but in most patients
   with late stages of cancer, immune cells may lose the ability to
   recognize tumor cells, how to activate their immune cells is a major
   problem, sodium glucosidate (SSG) is injected into ascites as a protein
   tyrosine phosphatase inhibitor to wake up immune cells and prepare for
   immunotherapy. We used single-cell RNA sequencing (scRNA-seq) to
   investigate whether and how SSG injected into ascites of pancreatic
   cancer elicits an immune response. Our study showed that the process of
   SSG fusion treatment of ascites and pleural effusion, the interaction
   between TandNK cells, MPs cells, monocytes and neutrophils was induced,
   and large numbers of genes were expressed, resulting in upregulation of
   immune response, which also approved that SSG is not only used as a
   protein tyrosine phosphatase inhibitor, but also it works as a protein
   tyrosine phosphatase inhibitor. It can also be used to regulate immune
   cell function, recruiting immune cells to the right place with the help
   of PD-1 or PD-L1 to fight cancer cells in ascites and pleural effusions
   in cancer patients.

   Keywords: sodium stibogluconate, immunotherapy with sodium
   stibogluconate, protein tyrosine phosphatase inhibitor, immune inducer,
   immune booster

Introduction

   The incidence and death rates for pancreatic cancer have been gradually
   increasing while the incidence and death rates for other common cancers
   have been declining. Only about 4% of pancreatic cancer patients live
   five years after diagnosis. For some patients, malignancies are only
   located to the pancreas without metastasis and their survival rates are
   higher due to surgical removal. Unfortunately, 80-85% of patients have
   advanced, unresectable cancer. In addition, pancreatic cancer does not
   respond well to most chemotherapy drugs ([33]1). We have recently
   experimented the injection of compound chemotherapy drugs plus hapten
   into advanced pancreatic cancer, which killed new tumors and induced an
   immune response, thereby awakening immune cells and prolonging the life
   of patients ([34]2). For advanced pancreatic cancer patients with
   pleural metastasis and pleural effusion, there are no good therapies
   and drugs to choose from, even the advanced immunotherapy will not be
   helpful ([35]3).

   Ascites and pleural effusion are recognized as a severe complication of
   pancreatic disease. Drug treatment for these conditions is limited by
   high costs, long hospital stays and failure rates; Invasive procedure
   is accompanied with a high morbidity and mortality rate. Endoscopic
   therapy is often the first-line treatment of pancreatic ascites and
   pleural effusion. The intervention is a 5 mm pancreatic sphincterotomy
   and endoscopic placement of a 7 Fr pancreatic stent, through which
   bridges the pancreatic ductal leak. The method offers a higher rate of
   success and provides a good treatment option for patients with
   pancreatic ascites and pleural effusion ([36]3, [37]4).

   However, it is still a form of physical therapy and can only provide
   relief. Thoracic infusion of chemotherapy drugs is also commonly used
   for the treatment of pancreatic cancer patients with ascites and
   pleural effusion, but the side effects are more severe that renders the
   method less effective, thus limiting its use. This paper will discuss
   the application of non-chemotherapy drug perfusion in pleural effusion,
   recruit and wake up the aggregation of immune cells, and use
   immunotherapy to provide an opportunity for the immunotherapy of
   pancreatic ascites and pleural effusion.

   In this study, we aimed to determine whether sodium stibogluconate
   (SSG) was used for pleural effusion infusion to effectively control
   pancreatic cancer ascites and pleural effusion and awake up various
   immune cells and recruit them to pleural effusion for therapeutic
   effect. SSG was commonly used for treatment of visceral leishmaniasis
   in a population with a high prevalence of HIV infection in Ethiopia and
   intralesional sodium stibogluconate for the treatment of localized
   cutaneous leishmaniasis at Boru Meda general hospital ([38]5, [39]6).
   SSG was injected with single drug into pleural effusion, pleural fluid
   samples were taken before and after treatment of SSG injection into
   pleural fluid. Then we used single-cell RNA sequencing (scRNA-Seq) to
   obtain transcriptome profiles of a total of 42249 EpithelialCells, and
   the proportion of changes in immune cells before and after treatment.
   Through comparative analysis of different samples of CIN and ascite
   samples, we comprehensively described the expression characteristics of
   malignant epithelial cells and immune cells, including Epithelial
   Cells, Ecs, Fibroblasts, Mural Cells, Tcells, Bcells, TandNK
   Neutrophils, Mast Cells, MPs, and Platelets, as well as the dynamic
   changes in cell percentage and cell subtype heterogeneity. Our results
   provide evidence that pleural effusion infusion was treated with single
   drug of SSG for induce acute immune response in pancreatic cancer
   ascites and pleural effusion, recruit and awaken immune cell for
   fighting cancer cells in the pleural fluid of pancreatic cancer.

Materials and methods

Ethical statement

   All procedures and protocols in the study have been reviewed and
   approved by the Ethical Committee of the Shandong Baofa Cancer
   Institute (TMBF 0010, 2021). All informed consent forms from patients
   have been signed prior to the start of the study.

Clinical specimens

   Pancreatic head cancer with retroperitoneal lymph node metastasis and
   liver metastasis. The tumor marker CEA was significantly elevated, the
   vital signs were stable, the skin and mucosa of the whole body had no
   yellow stain and bleeding points, the superficial lymph nodes were not
   swollen, the chest wall metastases were not detected, and there was a
   large amount of pleural fluid. It is not suitable for treatment of
   surgery and chemotherapy, and only palliative care can alleviate pain
   and prolong life. This experimental treatment was approved by the
   hospital ethics committee (TMBF 0010, 2015) in accordance with relevant
   guidelines and regulations.

   Pleural infusion was injected with 5.7g of Sodium stibogluconate (3
   sticks) once, once every other day, twice in total, and no adverse
   reactions such as fever and bleeding were observed. Ascite samples
   (25-30 ml) were taken before the treatment as control and after
   treatment as the treated samples for scRNA-Seq analysis, each samples
   contained over 10,000 cells. A total of three samples from the patient
   was taken as well at before and 4 days and 8 days after injection of
   Sodium stibogluconate.

Tissue disassociation and cells collection

   After sample extraction ([40] Figures 1C, D ), the fresh cells samples
   were immediately stored in the sCelLiVE® Tissue Preservation Solution
   (Singleron) on ice. The sample of ascites were transferred to a 15-ml
   centrifuge tube. The samples were then filtered with 40 µm sterile
   strainers, and centrifuged at 1,000 rpm at 4°C for 5 min. Next, 2 ml
   GEXSCOPE® red blood cell lysis buffer (RCLB, Singleron) was added to
   lyse the red blood cells for 10 min. Finally, the single cell
   suspension was collected after re-suspension with PBS, and trypan blue
   (Sigma) staining was used to calculate cell activity and cell count
   under a microscope ([41]7).

Figure 1.

   [42]Figure 1
   [43]Open in a new tab

   (A) Cell Type Reduction Map Display (UMAP). (B) Histogram of the
   proportion of cell types in different groups. (C) Display the
   dimensionality reduction map of major cell types in groups (UMAP, in
   the order of PC_B, PC_A1, PC_A2). (D) Violin map of marer genes in
   different cell types. (E) top10 heat maps of different cell types.

Single-cell RNA sequencing

   Single-cell suspensions (1~3×105 cells/mL) in PBS (HyClone) were loaded
   onto microwell chip using the Singleron Matrix® Single Cell Processing
   System. Briefly, the scRNA-seq library was constructed using the
   GEXSCOPE® Single Cell RNA Library Kits (Singleron). The library was
   lastly sequenced with 150 bp that was diluted to 4nM and paired-end
   reads on the IlluminaHiSeq X platform following an established protocol
   ([44]7). Sequencing data processing and quality control was performed
   as described in previous publications ([45]8, [46]9).

Data processing and analysis

   To identify differentially expressed genes (DEGs), genes expressed in
   more than 10% of the cells were selected in both groups of cells and
   with an average log (fold changes) value greater than 1 as DEGs.

   The cell type identity of each cluster was determined with the
   expression of canonical markers found in the DEGs using SynEcoSys
   database (Singleron Biotechnologies).

   The InferCNV package was used to detect the CNAs in malignant cells.
   Non-malignant cells (T and NK cells) were used as control references to