A novel mouse model for liver metastasis of prostate cancer reveals dynamic tumour‐immune cell communication

Abstract Objectives In contrast to extensive studies on bone metastasis in advanced prostate cancer (PCa), liver metastasis has been under‐researched so far. In order to decipher molecular and cellular mechanisms underpinning liver metastasis of advanced PCa, we develop a rapid and immune sufficient mouse model for liver metastasis of PCa via orthotopic injection of organoids from PbCre+; rb1f/f;p53f/f mice. Materials and Methods PbCre+;rb1f/f;p53f/f and PbCre+;ptenf/f;p53f/f mice were used to generate PCa organoid cultures in vitro. Immune sufficient liver metastasis models were established via orthotopic transplantation of organoids into the prostate of C57BL/6 mice. Immunofluorescent and immunohistochemical staining were performed to characterize the lineage profile in primary tumour and organoid‐derived tumour (ODT). The growth of niche‐labelling reporter infected ODT can be visualized by bioluminescent imaging system. Immune cells that communicated with tumour cells in the liver metastatic niche were determined by flow cytometry. Results A PCa liver metastasis model with full penetrance is established in immune‐intact mouse. This model reconstitutes the histological and lineage features of original tumours and reveals dynamic tumour‐immune cell communication in liver metastatic foci. Our results suggest that a lack of CD8+ T cell and an enrichment of CD163+ M2‐like macrophage as well as PD1+CD4+ T cell contribute to an immuno‐suppressive microenvironment of PCa liver metastasis. Conclusions Our model can be served as a reliable tool for analysis of the molecular pathogenesis and tumour‐immune cell crosstalk in liver metastasis of PCa, and might be used as a valuable in vivo model for therapy development.


| INTRODUC TI ON
Metastasis is the major cause of prostate cancer (PCa)-related death. 1 Bone metastasis, as one of the most common metastatic sites in advanced PCa, has been extensively investigated over the past decades. 2 Accumulating evidence has demonstrated that liver is a frequently afflicted site in advanced PCa, especially in disease relapse after treatment of potent androgen receptor pathway inhibitors (ARPI). 3 In particular, it has been reported by independent groups that a large portion of ARPI-induced neuroendocrine prostate cancer (NEPC), an aggressive variant of castration-resistant PCa, is associated with liver metastasis. 4,5 Currently, scarce treatment options such as chemotherapy for those patients often achieve poor responses. In spite of the daunting prognosis of PCa liver metastasis, the cellular and molecular mechanisms underlying PCa liver metastasis have been severely under-researched so far.
Unlike bone metastasis that usually cause severe notable pain in PCa patients, 6 liver metastasis is often symptomless at the beginning. Thus, when liver metastasis is clinically detected, many patients have reached the end stage of their disease. 7 These clinical features make it difficult to collect liver metastatic samples from PCa patients for analysis. Current available genetically engineered mouse models (GEMM) or patient-derived cell lines or xenografts fail to reconstitute the highly penetrant liver metastasis. Due to these long-standing barriers and limitations, a stable, rapid and immune sufficient model of PCa liver metastasis is in urgent need to facilitate in-depth mechanism investigations and new therapy development or drug screening.
When colonized in the liver, tumour cells encountered and communicated with local resident cells, especially immune cells, in the metastatic niche. 8 A tumour supportive microenvironment in distal organs is required for the survival and growth of metastatic tumour cells. 9 Although the 'seed-and-soil' theory can provide reasonable explanations for tumour metastasis, 10  and PbCre + ;pten f/f ;rb1 f/f (pten Δ/Δ rb1 Δ/Δ ) were currently used to study metastatic PCa. [11][12][13][14] These models had been well characterized by independent groups. For example, pten Δ/Δ p53 Δ/Δ model revealed admixed adenocarcinoma and sarcomatoid histological feature, frequent lymph node metastasis and occasional lung metastasis. 11 In contrast, rb1 Δ/Δ p53 Δ/Δ and pten Δ/Δ rb1 Δ/Δ displayed prominent neuroendocrine differentiation and metastases in common sites including lymph node, lung, bone and liver. 12,13 Here, we utilize tumour organoids from rb1 Δ/Δ p53 Δ/Δ and pten Δ/Δ p53 Δ/Δ GEMMs and generate a stable and rapid liver metastatic PCa mouse model with a high penetrance in immune-sufficient mice. Using a mCherry + niche-labelling reporter system, dynamic tumour-immune cell communications at different metastatic stage in the liver are depicted.

| Orthotopic inoculation in the murine prostate
MCherry + niche-labelling lentivirus infected organoids were injected into the anterior lobe of prostate of 6-or 8-week-old C57BL/6 mice with around 2,400 organoids for each mouse. Organoids were dissociated with 0.25% trypsin-EDTA at 37°C for 1 minute. The organoid suspension was then mixed with Matrigel at 1:1 (v/v) ratio. Mice were anaesthetized using continuous flow of 1% isoflurane. The lower abdomen was sterilized with chlorhexidine iodine and a transverse incision of 1cm in length was made. 50 µL of organoid suspension was slowly injected into the anterior prostatic lobe where a bubble was clearly identified after inoculation. After inoculation, prostates were carefully put back into the body cavity, and the muscle and skin layer were then carefully sutured. Surgery was performed on a heating pad until mice completely recovered from anaesthesia.
Other detailed materials and protocols can be accessed in Supplemental Methods and Materials.
We then generated organoid lines from these two PCa models using a previously reported method, 16 which allowed easy expansion and manipulation of organoids in vitro. The knockout efficiency of pten, p53 and rb1 in organoid lines were confirmed by qRT-PCR after a long-term culturing (Supplemental Figure 1C). As shown in Figure 1D,E, unlike the typical WT prostate organoids with round shape and regular sphere, pten Δ/Δ p53 Δ/Δ tumour organoids exhibited malignant phenotypes including evident nuclear atypia, dense spheres and intruding pseudopodium. In contrast, rb1 Δ/Δ p53 Δ/Δ GEMM tumour organoids were featured with smaller cell size, with sheets or nest-like histological structure. IF staining of androgen receptor (AR), basal cell maker p63, luminal cell marker K8 and neuroendocrine maker Syp were further performed in these two lines of organoids. As shown in Figure 1F,G, we found a coexistence of AR + and p63 + tumour cells in most of pten Δ/Δ p53 Δ/Δ GEMM tumour organoids ( Figure 1F), but Syp + cells were hardly detected. In contrast, rb1 Δ/Δ p53 Δ/Δ GEMM organoids contained a high percentage of Syp + cells but expresses low levels of K8, AR and p63 ( Figure 1G).
We found that rb1 Δ/Δ p53 Δ/Δ GEMM organoids exhibited decreased expressions of Ar and probasin (Pbsn) but increased neuroendocrine marker Chga at mRNA levels (Supplemental Figure 1C). Collectively, these results suggest that the two lines of organoids in vitro stably sustain specific cell lineage signatures of their parental primary tumours in vivo.  Figure 2B). Of note, both AR + /K8 + adenocarcinoma and p63 + /K5 + squamous-like basal cell carcinoma were observed within a same section of pten Δ/Δ p53 Δ/Δ ODT (Supplemental Figure 2C), in consistent with the primary tumour from pten Δ/Δ p53 Δ/Δ GEMMs. Immunohistochemistry (IHC) staining suggested that rb1 Δ/ Δ p53 Δ/Δ ODT expressed high level of neuroendocrine markers including Syp and Ncam. In contrast, luminal markers, such as AR and K8, and basal markers p63 were hardly detected in the rb1 Δ/Δ p53 Δ/Δ ODT section. These results suggested a similar histological feature and lineage signature between ODTs and their original GEMM primary tumours in both lines ( Figure 2D).

| Rb1 Δ/Δ p53 Δ/Δ ODT drives liver metastasis with full penetrance and high efficiency
Next, distal metastases were investigated in both pten Δ/Δ p53 Δ/Δ and rb1 Δ/Δ p53 Δ/Δ organoid-implanted mice. Similar to pten Δ/Δ p53 Δ/Δ GEMMs, pten Δ/Δ p53 Δ/Δ organoid-implanted mice did not display significant distal metastases. In contrast, rb1 Δ/Δ p53 Δ/Δ organoidimplanted mice rapidly developed distal metastases, in particular, a significant liver (20/20) metastasis with full penetrance ( Figure 2E). Strikingly, rb1 Δ/Δ p53 Δ/Δ ODTs were capable to initiate liver metastasis as early as 20 ~ 30 days after implantation, compared to 28 weeks in rb1 Δ/Δ p53 Δ/Δ GEMM model for liver metastasis to be visible. Before the mice were succumbed, we found that the sizes of rb1 Δ/Δ p53 Δ/Δ ODT-induced liver metastatic foci were significantly larger than those in the liver of rb1 Δ/Δ p53 Δ/Δ GEMMs (Supplemental Figure 3A,B). Moreover, the number of liver metastatic foci per lobe in rb1 Δ/Δ p53 Δ/Δ organoid-implanted mice was dramatically higher than that of rb1 Δ/Δ p53 Δ/Δ GEMM counterparts (Supplemental Figure 3C). In order to exclude the possibility that orthotopic inoculation may introduce disruptions to the prostate gland or vasculature and lead to dissemination, fluorescent DiD- Due to the massive liver metastasis, the liver weight of rb1 Δ/ Δ p53 Δ/Δ organoid-implanted mice were higher than normal livers from age-matched mice (Supplemental Figure 3E). It's noteworthy that rb1 Δ/Δ p53 Δ/Δ organoid-implanted mice had a significantly shorter median life span of 45 days and all died by 60 days after orthotopic inoculation, compared to 210 days of median life span in rb1 Δ/Δ p53 Δ/Δ GEMM ( Figure 2F). We further compared the liver metastatic frequencies of intact organoids and single cell suspensions generated from fully digested organoids after orthotopic inoculation. As shown in Supplemental Figure 3F,G, those fully digested organoids showed significantly lower amount of liver metastasis as compared to intact organoids. These data suggested that intact cell-cell communication in 3D organoid culture system was required for a high efficiency of liver metastasis. Moreover, we carefully evaluated the liver metastatic potential of these rb1 Δ/Δ p53 Δ/Δ organoids during long-term culturing, freezing, thawing and serial passaging. No evident alterations were found in either the morphological feature (Supplemental   Figure 4B). The amount of sLP-mCherry fluorescent protein 'swallowed' by GFP + 293T were determined in a non-contact or a contact manner using flow cytometric analysis. As shown in Figure 4C, GFP + recipient 293T cells uptook significantly greater amount of mCherry proteins in a directly contacted cocultured system (16.6%) than that treated with conditioned-medium (CM) (2.62%). These results suggested that cell-cell contact contributed to a higher lipid permeable sLP-mCherry uptake, and this mCherry-Luc reporter was able to efficiently label niche cells.
Next, mCherry-Luc reporter lentivirus infected rb1 Δ/Δ p53 Δ/Δ organoids were orthotopically inoculated in the prostate of WT C57BL/6 mice in order to directly study the cell communications between immune cells uptaking mCherry-Luc and tumour cells. As shown in Figure 4D, the rapid outgrowth of rb1 Δ/Δ p53 Δ/Δ ODT in the prostate was clearly monitored by an IVIS bioluminescent imaging system. According to our observations, evident but weak bioluminescent signal in the lower abdomen could be detected at 20 ~ 30 days after orthotopic inoculation. Since liver metastasis could be detected by microscopic inspection at this stage ( Figure 4E), we defined this F I G U R E 3 Establishment of a rapid liver metastasis model for PCa in immunesufficient mice using rb1 Δ/Δ p53 Δ/Δ GEMM organoids. A-B, H&E and IHC staining images show the metastatic tumor foci in a liver lobe of rb1 Δ/Δ p53 Δ/Δ GEMM mice (A) and rb1 Δ/Δ p53 Δ/Δ GEMM organoids implanted mice (B). The metastatic region (meta, green), the border between the metastasis and the hepatocytes (border, blue) and the hepatic region (hepa, red) in the liver sections are shown. Ki67 antibody stains the proliferating cells of the tumor foci. Albumin is used as a marker of hepatocytes. AR antibody labels the tumor cells originated from the PCa. Syp and Ncam are used as markers for PCa cells with neuroendocrine differentiation phenotype period as the early stage. Around 30 ~ 40 days, bioluminescent intensity and H&E staining ( Figure 4F) clearly showed the sporadic liver metastases at this stage ( Figure 4E). We termed this period from 30 to 40 days as the middle stage. At 50 to 60 days, the intensity and region of bioluminescence expanded further with a prominent signal in the liver (pinpointed with red arrows). Accordingly, we termed this period from 50 to 60 days as the late stage of liver metastasis. As shown in Figure 4F, the primary prostate tumour, liver and lung from a rb1 Δ/Δ p53 Δ/Δ ODT implanted mouse at 50 days were  Figure 4E, no significant difference in tumour weights was detected between these two groups. And no significant differences were found in immune cell infiltration including macrophages, Figure 4F,G), suggesting that mCherry-Luc niche-labelling reporter F I G U R E 4 A niche-labeling system to reveal tumor-niche communications in the liver metastasis of rb1 Δ/Δ p53 Δ/Δ ODT mouse model. A, A lentiviral construction containing the PKG promoter driven transactivator of transcription (TATk) peptide followed by a secreted mCherry protein and a bioluminescent luciferase cassette. B, Proof of concept experiment of the niche labeling system using conditioned-medium (CM) or directly contacted co-culture method of 293T cells. C, Flow cytometry results show that the fluorescent mCherry protein can be secreted by mCherry + 293T cells and uptaken by the GFP + 293T cells. GFP + 293T cells were cultured by transferring-CM of mCherry + 293T cells or directly co-cultured with mCherry + 293T cells in a contact manner. D, The bioluminescent images of rb1 Δ/Δ p53 Δ/Δ ODT mice at early (20 d after inoculation), middle (35 d after inoculation) and late stages (50 d after inoculation). The liver metastatic tumor foci are indicated by red arrows. Rb1 Δ/ Δ p53 Δ/Δ GEMM organoids are infected with the niche-labeling lentiviruses and orthotopically inoculated in the prostate of WT C57BL/6 mice (8-weekold). E, H&E staining shows the liver metastatic foci in liver at early, middle, and late stages. Scale bar = 4 mm. F, The bioluminescent images and bright field images of PCa, liver and lung isolated from rb1 Δ/Δ p53 Δ/Δ ODT mice did not introduce profound immunogenic effect at least in local prostatic microenvironment. Collectively, our results suggested that this niche-labelling reporter was capable to vividly reflect both the status of primary tumour and the liver metastasis at different stages.

| Depiction of the niche-labelled immune cells in the liver metastasis of rb1 Δ/Δ p53 Δ/Δ ODT model
In order to reveal the immune cell composition that uptook mCherry protein secreted by tumour cells in the liver metastatic niche, the liver was cut into pieces and generated into single cell suspension (Supplemental Figure 5A). Next, we analysed the mCherry and CD45 double positive (mCherry + CD45 + ) cell population by flow cytometry (Figure 5A). Following the gating strategies in Supplemental Figure 5B,C, we deciphered the dynamic changes of macrophage (F4/80 + CD11b + ), neutrophil (Ly6G + CD11b + ), dentritic cell (DC, CD11c + ), B cell (CD19 + ) and T cell (CD3 + ) in mCherry + CD45 + niche-labelled immune cells at early, middle and late stages of liver metastasis ( Figure 5B). Macrophage ( Figure 5C) and neutrophil ( Figure 5D) were the most abundant fractions in tumour-communicating immune cells in liver metastasis.

F I G U R E 5
Immune-suppressive CD163 + macrophages are significantly increased in the metastatic niche at the late stage of PCa liver metastasis. A, Flow cytometric analysis of immune cells that are mCherry + in the liver metastasis of rb1 Δ/Δ p53 Δ/Δ ODT. B, A schematic of immune cells examined in the liver metastatic niche. C-F, Representative flow cytometric plots and quantification of the ratio of macrophages (C), neutrophils (D) and dentritic cells (DCs) (E) in CD45 + mCherry + cells, and M1, M2 macrophages (F) in CD45 + mCherry + macrophages in rb1 Δ/Δ p53 Δ/Δ ODT liver metastasis niche at the early, middle and late stages. Unpaired two-tailed Student's t-test is used for statistical analysis. The data are presented as the means ± SEMs. *P < .05, **P < .01, ***P < .001, ****P < .0001, n = 5 Macrophages accounted for 46.43% of total CD45 + mCherry + immune cells at the early stage, then dropped to 25.4% at the middle stage, and eventually rebounded to 51.93% at the late stage ( Figure 5C). Distinct from macrophages, the ratio of neutrophils in CD45 + mCherry + immune cells steadily increased from 17.6% at the early stage to 20.3% at the middle stage, then reached a peak value of 27.2% at the late stage ( Figure 5D). Unlike macrophage and neutrophil, DC was relatively rare in mCherry labelled immune cells. As shown in Figure 5E, there was a slight increase in DC from early stage (3.05%) to middle stage (4.65%), then the ratio of DC dropped again (3.43%) as it came to the late stage.
It is well accepted that macrophage can be divided into M0, M1 and M2 polarized subsets. 18 It is proposed that M2 macrophage displayed tumour-permissive activity while M1 macrophage exhibited pro-inflammatory functions. 19 To characterize the phenotype of the mCherry + macrophages at different stages in the metastatic niche, CD86 and CD163 were used to distinct M1 and M2 macrophage, respectively. 20 As shown in Figure 5F, over 65% of macrophages were positive for CD163 expression, suggesting that M2 like macrophages accounted for the predominant subset in the mCherry + macrophages. Moreover, the ratio of CD163 + M2 macrophage initially decreased from 80.1% at the early stage to 66.5% at the middle stage. Eventually, the ratio rebounded to 83% as it came to the late stage. Collectively, our results revealed that macrophage and neutrophil were the most sufficient immune cell subsets that interacted with invading tumour cells in the liver metastases. The predominant subset of M2-like macrophages reached a peak value at the late stage, suggesting that an immune-suppressive microenvironment was created in the liver metastatic niche.

| A paucity of CD8 + T cells and abundant PD1 + CD4 + T cells contribute to an immunesuppressive liver metastatic niche
We further assessed the ratio of mCherry labelled B cells and T cells across different stages of liver metastasis (Supplemental Figure 5C). Figure 6A, the subset of B cells was rare in mCherry labelled immune cells. It slightly increased from 2.18% at the early stage to 2.56% at the middle stage, and finally reached to 3.46% at the late stage. In contrast, the number of mCherry labelled CD3 + T cell was more abundant as compared to B cells. CD3 + T cells accounted for around 16.7% of mCherry + immune cells at the early stage, then dropped to 9.78% at middle stage, and finally went up to 12.1% at the late stage ( Figure 6B). Of note, the cytotoxic CD8 + T cells were very rare in mCherry + T cells, which ranged from 0.20% to 3.55% across different stages ( Figure 6C). These results suggested that the rapid progression of liver metastasis was associated with a lack of cytotoxic CD8 + T cells in liver metastatic niche. In contrast to CD8 + T cells, the ratio of CD4 + T cells initially decreased from 7.57% at the early stage to 5.08% at the middle stage. Finally, the ratio of CD4 + T cells rebounded to 9.21% at the end stage ( Figure 6D). Therefore, there was a negative co-relation between CD8 + T and CD4 + T cells across different stages.

As shown in
We also assessed the expression of immune checkpoint protein PD1 on the surface of T cells. 21 As shown in Figure 6E, flow cytometric analysis indicated that the PD1 + CD4 + T cells initially decreased from 68% at the early stage to 42.8% at the middle stage. Intriguingly, the ratio of PD1 + CD4 + T cells increased to 81% at the late stage.
These results suggested that a lack of CD8 + T cells and abundance in PD1 + CD4 + T cells further generate an immune-suppressive liver metastatic niche.

| D ISCUSS I ON
In the current study, we developed a stable and rapid liver metasta-  Figure 7, using a mCherry-Luc niche-labelling reporter, we revealed that the most abundant immune cells in response to tumour cells in liver were macrophages, which were predominantly featured with an M2 phenotype. Dynamic tumour-immune cell communication was depicted at different stages in the liver metastatic niche.
The paucity of CD8 + T cells and increased number of PD1 + CD4 + T cells might contribute to an immune-suppressive microenvironment that supported the tumour cell colonization in the liver.
In the rb1 Δ/Δ p53 Δ/Δ ODT model, liver metastases are initiated with a full penetrance as early as 20 ~ 30 days. The traditional approach to generate metastatic PCa GEMM requires time-and labourconsuming way to cross multiple genetically modified mouse lines.
In addition, the liver metastasis develops with a low efficiency, takes a long period of time and is highly variable among individual animals.
Up to now, there is no ideal in vivo model for liver metastasis using patient-derived cell lines or xenografts to the best of our knowledge.
Existing liver metastasis models which use intracardiac injection, 22 hepatic inoculation 23 or ectopically splenic implantation 24  to the liver. 25 In our current work, we carefully compare two frequently used PCa mouse models including rb1 Δ/Δ p53 Δ/Δ GEMM and pten Δ/Δ p53 Δ/Δ GEMM. We find that co-inactivation of rb1 and p53 exhibited liver metastasis with a higher efficiency. However, we do not detect significant liver metastasis in pten Δ/Δ p53 Δ/Δ GEMM even at the very end stage of their diseases. A recent literature reported that Rb1 and p53 cooperatively suppress cell lineage plasticity and prevent the transdifferentiation from AR dependent prostate adenocarcinoma to NEPC. 12 Co-inactivation of rb1 and p53 in the murine prostate confers a significant neuroendocrine feature and resistance to androgen deprivation. 13 In contrast, pten and p53 mutant PCa display a mixed adenocarcinoma with occasional squamous tumour features.
This discrepancy in rb1 Δ/Δ p53 Δ/Δ GEMM and pten Δ/Δ p53 Δ/Δ GEMM models suggest that distinct driver mutations in PCa elite differential molecular programmes and result in different metastasis potential and site preference. Although the cause-and-effect relationship remains further investigations, our findings indicated that Rb1 might be a critical gatekeeper that suffices liver metastasis in advanced PCa. F I G U R E 6 A paucity of CD8 + T cells and abundant PD1 + CD4 + T cells further contribute to the immunesuppressive liver metastatic niche in rb1 Δ/Δ p53 Δ/Δ ODT. A-E, Representative flow cytometric analysis and quantification of the ratio of B cells (A), CD3 + T cells (B), CD8 + T cells (C) and CD4 + T cells (D) in CD45 + mCherry + cells, and PD1 + CD4 + T cells (E) in CD45 + mCherry + CD4 + T cells in rb1 Δ/Δ p53 Δ/Δ ODT liver metastasis niche at the early, middle, and late stages. Unpaired twotailed Student's t-test is used for statistical analysis. The data are presented as the means ± SEMs. *P < .05, **P < .01, n = 5 Once spread to the liver, an immune-suppressive microenvironment is highly required for the survival and growth of metastatic tumour cells in the metastatic niche. Due to the difficulty in collecting clinical liver metastasis specimens and the lack of a reliable liver metastasis mouse model, the tumour-immune cell communication in the liver metastatic niche remains largely unknown. We introduce a mCherry + niche-labelling lentiviral reporter system to the rb1 Δ/Δ p53 Δ/Δ ODT model. Herein, this niche-labelling reporter has at least uncovered two major interesting findings. First, the majority of immune cells that takes up the mCherry protein is CD163 + M2 like macrophage. Second, insufficient CD8 + T cells and abundant PD1 + CD4 + T cells that react to invading tumour cells might contribute to regional immune suppression in the liver metastatic niche.
Interestingly, during the preparation of our manuscript, a very recent paper reported that liver metastases were capable to siphon activated Fas + CD8 + T cells from peripheral blood to the liver, where these cytotoxic T cells were eliminated by FasL + CD11b + F4/80 + macrophages. 26 This is in line with our result of an inverse association between the ratio CD8 + T cells and M2-like macrophages across early, middle and late stages in the liver metastatic niche of PCa.
Our findings highlight a requirement of an immune-suppressive environment and reveal the key immune cell component for PCa cell outgrowth in liver metastasis.
Up till now, a few of methods including intracardiac injection 27 and hemi-spleen injection 28 has been introduced to study liver metastasis in different cancer types. However, intracardiac and hemi-spleen injections fail to mimic the tumour progression in the orthotopic sites where the primary tumour develops and skip the epithelia-mesenchymal transition (EMT) and intravasation steps during metastasis. Here, we successfully established a stable, rapid and immune sufficient mouse model for PCa liver metastasis, which mimics the entire navigation of tumour metastasis from local site to the liver. Therefore, our model can be used as a powerful tool for to study cellular and molecular mechanisms of liver metastasis.
Compared to GEMMs and patient-derived cell lines or xenografts, it is time-and labour-friendly and harbours intact immune system. It can serve as a valuable platform for novel therapy development and large-scale screening of drugs for the treatment of metastatic PCa. F I G U R E 7 Schematic graph of the dynamic tumor-immune cell communication in the liver metastatic niche of the rb1 Δ/Δ p53 Δ/Δ ODT mouse model. A, A schematic model showing a dynamic change of CD8 + mCherry + T cells, PD1 + CD4 + mCherry + T cells, and CD163 + M2 macrophages at the early, middle and late stages of liver metastasis in the rb1 Δ/ Δ p53 Δ/Δ ODT model

ACK N OWLED G EM ENTS
The study was supported by funds to HH Zhu from the National Key

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data of this study can be obtained from the corresponding authors when requested reasonably.